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Introduction: 2-[ 18 F] Fluoro-d-deoxyglucose (FDG) positron emission tomography (PET) is a relevant diagnostic procedure for staging lung cancer. However, accurate evaluation of lymph node metastases by PET is controversial because of false-positive FDG uptake., Patients and Methods: A total of 245 patients with lung cancer were retrospectively analyzed. Standardized maximum uptake values (SUV max ) of the primary tumor and lymph nodes were compared to pathologic lymph node metastases to correlate PET findings with clinicopathologic variables and patient outcomes., Results: The SUV max values of metastatic lymph nodes were significantly higher than those of lymph nodes without metastases (P = .0036). When SUV max  ≥ 4 was defined as PET positive for metastasis, the sensitivity, specificity, and accuracy were 48.1%, 79.8%, and 73.1%, respectively. Multivariate logistic regression analysis showed that age > 75 years, bilateral hilar FDG uptake, and no lymph node swelling were significant factors related to false-positive lymph node metastases. Smoking status, FDG uptake in the primary tumor, and concurrent lung diseases were not significant factors., Conclusion: Metastatic lymph nodes show higher FDG uptake than false-positive lymph nodes, and older patient age, bilateral hilar FDG uptake, and no swollen nodes are associated with no metastases. Patients with lymph node metastases have worse survival than those with false-positive FDG-PET findings. However, abnormal FDG uptake in the lymph node is an important prognostic factor., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Activating transcription factor 5 (ATF5) is a member of the ATF/cAMP response element-binding protein family. Our research group recently revealed that ATF5 expression increases the invasiveness of human lung carcinoma cells. However, the effects of ATF5 on the invasive potential of other cancer cells lines remain unclear. Therefore, in this study, we investigated the role of ATF5 in the invasive activity of diverse human cancer cell lines. Invasiveness was assessed using Matrigel invasion assays. ATF5 knockdown resulted in decreased invasiveness in seven of eight cancer cell lines tested. These results suggest that ATF5 promotes invasiveness in several cancer cell lines. Furthermore, the roles of ATF5 in the invasiveness were evaluated in three-dimensional (3D) culture conditions. In 3D collagen gel, HT-1080 and MDA-MB-231 cells exhibited high invasiveness, with spindle morphology and high invasion speed. In both cell lines, knockdown of ATF5 resulted in rounded morphology and decreased invasion speed. Next, we showed that ATF5 induced integrin-α2 and integrin-β1 expression and that the depletion of integrin-α2 or integrin-β1 resulted in round morphology and decreased invasion speed. Our results suggest that ATF5 promotes invasion by inducing the expression of integrin-α2 and integrin-β1 in several human cancer cell lines., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Nucleocapsid protein of HIV, containing two CCHC-type zinc fingers connected by a linker, is a multi-functional protein involved in many critical steps of the HIV life cycle. Several in vitro investigations demonstrated that the reactivities of the first zinc finger flanked by the linker of HIV-1 NCp7 and HIV-2 NCp8 were essential for binding to viral RNA, however, that of the second zinc finger flanked by the linker of NCp7 was very weak and non-specific, whereas the part of NCp8 called NCp8-f2, interacted strongly and specifically with viral RNA. In this study, the three-dimensional structure of NCp8-f2 was determined for the first time. Furthermore, we established that NCp8-f2 specifically binds to the stem-loop SD in viral RNA, and that the hydrophobic cleft and the basic residues close to the cleft were essential for specific binding to SD. We discuss the functional significance of NCp8-f2 for NCp8 being a multi-functional protein.

Background: We and other researchers have previously reported that pulmonary adenocarcinomas with epidermal growth factor receptor (EGFR) mutations are usually sensitive to gefitinib, an EGFR-specific tyrosine kinase inhibitor, although this relationship is not complete. In this study, we searched for mutations or changes in the expression of genes downstream to EGFR and evaluated their relationship with the effectiveness of gefitinib., Methods: We studied 78 lung cancer patients who had recurrent disease after surgical resection and were treated with gefitinib. We searched for mutations occurring in the KRAS and PIK3CA gene. We also evaluated the expression level of EGFR, PIK3CA, and PTEN by real-time reverse transcriptase polymerase chain reaction. Gefitinib effectiveness was evaluated by imaging studies; a survival analysis was also done., Results: We found seven (9%) somatic mutations in KRAS and two (2%) in PIK3CA. EGFR mutations were present in 44 (56%). KRAS mutations were found only in tumors without EGFR mutations, whereas PIK3CA mutation was found in tumors with EGFR mutation. Tumor response was assessable in 52 tumors. None of the six tumors with KRAS mutations responded to gefitinib treatment; however, two tumors with PIK3CA mutations showed partial response. Survival was significantly longer in patients with EGFR mutations or in those without KRAS mutations. In tumors with EGFR mutations, survival was longer in those with high PIK3CA or PTEN expression than in those with low expression of these molecules., Conclusions: An evaluation of the KRAS mutation, as well as PIK3CA and PTEN expression, might help identify lung cancer patients who are most suitable for gefitinib treatment.

We measured the accuracy of the continuous intra-vascular blood-gas monitoring system (Paratrend 7, PT7) placed in the jugular venous bulb in 18 adult patients having cardiac or aortic surgery with hypothermic cardiopulmonary bypass (CPB). After induction of anaesthesia, a PT7 sensor was inserted through a 20-gauge venous catheter into the right jugular venous bulb. Blood samples were drawn from the venous catheter and measured with a blood gas analyser (BGA). Five to eight paired measurements using the PT7 and blood samples were made per patient, and bias and precision were calculated for each patient using the Bland-Altman method. The ranges for the blood sample measurements were: pH 7.12 to 7.59, PCO(2) 3.7 to 9.6 kPa, PO(2) 3.5 to 16.0 kPa, oxygen saturation 40 to 99%, bicarbonate 18.6 to 34.4 mmol l(-1), and base excess -7.8 to 12.5 mmol l(-1). Bias and precision values were 0.014/0.071 for pH, 0/0.90 kPa for PCO(2), and -0.16/1.18 kPa for PO(2). These values were comparable with those previously made on arterial blood. However, precision for oxygen saturation in each patient varied 2.3 to 23.6% (95% CI: 6.3 to 12.9%), which was unsatisfactory for clinical measurements. Deep hypothermia ( approximately 19.6 degrees C) and marked haemodilution ( approximately 13.5%) during CPB did not influence the reliability of the PT7 sensor. Thus, we concluded that continuous intra-jugular venous blood-gas monitoring is clinically feasible using the PT7 and may provide valuable information during CPB.

Although epidermal growth factor (EGF) accelerates gastric mucin biosynthesis, information on whether its activation is limited to the specific mucus-producing cells is lacking. In this paper, we investigated the effects of EGF on mucin biosynthesis and the expression of its receptor in distinct layers of rat gastric mucosa, including the possible participation of nitric oxide (NO). EGF enhanced the incorporation of [3H]glucosamine and [14C]threonine into the mucin in the full-thickness tissues of the gastric mucosa. This stimulation disappeared on the removal treatment of the surface mucosal layer chiefly consisting of surface mucus cells. The EGF-induced increase in [3H]-labeled mucin in the full-thickness mucosa was not suppressed by either NG-nitro-L-arginine (10(-5) M) or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (10(-5) M). The EGF-receptor-mRNA expression was high in the surface mucosal layer but low in the deep and muscle layers of the stomach. These results suggest that EGF-induced stimulation of mucin biosynthesis is limited to the surface mucus cells of the rat gastric mucosa and is independent of the NO pathway.

Study Objective: To investigate the effects of nicardipine-, nitroglycerin-, and prostaglandine E1-induced hypotension on cerebrovascular carbon dioxide (CO2) reactivity over a wide range of arterial CO2 tension (PaCO2) (PaCO2; range 25 to 50 mmHg)., Design: Prospective, randomized study., Setting: Operating room of a university-affiliated hospital., Patients: 36 ASA physical status I and II patients without cerebrovascular disease, hypertension, or diabetes mellitus, undergoing an elective abdominal surgery., Interventions: Patients were randomly allocated to one of three groups (nicardipine-, nitroglycerin-, or prostaglandin E1-induced hypotension group; 12 in each group). Anesthesia was induced and maintained with a bolus dose, followed by a continuous infusion of propofol (6.7 +/- 1.5 mg/kg/hr) and fentanyl (1.68 +/- 0.4 micrograms/kg/hr). Deliberate hypotension of mean arterial pressure 55 to 60 mmHg was induced and maintained with a bolus dose, followed by a continuous infusion of nicardipine (6.80 +/- 0.75 micrograms/kg/min), nitroglycerin (3.20 +/- 1.10 micrograms/kg/min), or prostaglandin E1 (0.103 +/- 0.052 microgram/kg/min)., Measurements and Main Results: Time-averaged mean red blood cell velocity in the right middle cerebral artery (Vmca) at PaCO2 ranging from 25 to 50 mmHg was measured with transcranial Doppler ultrasonography. A minimum of six simultaneous measurements of Vmca and PaCO2 were obtained during baseline and deliberate hypotension in each patient. Absolute slope between Vmca and PaCO2 during baseline and deliberate hypotension was determined individually by linear regression analysis. Absolute slope was treated as the variable, because it yielded a significant close correlation coefficient (r > 0.95; p < 0.05). Comparisons between baseline and deliberate hypotension were made by analysis of variance for repeated measures. Mean absolute slope was significantly reduced from 1.88 +/- 0.57 cm/sec/mmHg (mean +/- SD) to 1.21 +/- 0.46 in the nicardipine group (p < 0.05), from 1.75 +/- 0.69 to 1.35 +/- 0.47 in the nitroglycerin group (p < 0.05), and from 1.95 +/- 0.89 to 1.33 +/- 0.70 (p < 0.05) in the prostaglandin E1 group, respectively., Conclusion: Nicardipine-, nitroglycerin-, and prostaglandin E1-induced hypotension attenuate the human cerebrovascular CO2 reactivity during propofol-fentanyl anesthesia.

A novel isoform of rat estrogen receptor (ER) beta, ER beta 2, which is a putative alternative splicing product of the reported ER beta (ER beta 1) has been identified. Rat ER beta 2 cDNA contains an additional, in-frame 54 base pair insertion in the ligand binding domain of ER beta 1, which generates an 18 amino acid residue insertion. Northern blot and RT-PCR analyses revealed that ER beta 2 coexists with ER alpha and ER beta 1 in all tissues examined including brain, lung, liver, kidney, fat, bone, uterus, prostate, and ovary. The insertion caused loss of ligand binding activity of ER beta 2, whereas the ability to bind the palindromic estrogen response element (ERE) was retained. In an ERE-containing luciferase reporter gene assay using COS-1 cells, ER beta 2 failed to activate estrogen-dependent transcription. Furthermore, ER beta 2 dose dependently suppressed the ER alpha- and ER beta 1-mediated transcriptional activation. These results suggest that rat ER beta 2 functions as a negative regulator of estrogen action.

We examined the effect of estrogen on mitogen-activated protein kinase (MAPK) in osteoblastic cells. Rat ROS 17/2.8 cells were exposed to 17beta-estradiol (E2) and MAPK activity in the cells was measured by an in vitro phosphorylation assay. E2 treatment caused a rapid and transient MAPK activation within 5 min. Insulin-like growth factor-I, which acts via their membrane receptors, caused a similar effect, but it required 10 min to reach the maximum level. Western blot analyses with anti-MAPK and anti-phosphotyrosine antibodies demonstrated that the E2 activation of MAPK was accompanied by phosphorylation of the enzyme. The concentration range (10 nM-1 pM) of E2 needed for this MAPK activation was less than that (1 microM-0.1 nM) needed for the transcriptional activation via the nuclear estrogen receptor (ER). These data provide the first evidence of MAPK activation by E2 through phosphorylation, which may be mediated through a putative plasma membrane receptor in the cultured bone cells.

It has been suggested that heterosegmentally activated slow positive potentials (HSP), recorded from the spinal cord of rat and humans, are feedback inhibitory potentials. The present study was carried out to define ascending and descending spinal tracts and the sites of central nuclei involved in the production of these HSP, and the effects of ketamine on these central nuclei. The spinal cords in ketamine-anaesthetized rats were transected to determine the ascending and descending tracts involved in the production of hindpaw (HP) and forepaw (FP) HSP, respectively. Lesions of the brain at various levels were performed stereotactically during ketamine anaesthesia. Dorsal one-third resection of the cord at the T8-9 level did not affect HSP significantly, while contralateral lesion of the dorsal two-thirds of the cord decreased FP-HSP but not HP-HSP during ketamine. Bilateral transection of the ventral one-third of the cord abolished both HSP. Ablation of the cerebral cortex, cerebellum, thalamus, midbrain and pons did not affect HSP significantly. However, transection of the middle medulla decreased, while transection of the most caudal part of the medulla completely abolished both HSP. Ketamine decreased HSP even in the medulla-spinal cord preparation and the segmental slow positive wave in spinalized animals. In ketamine-anaesthetized rats, ascending and descending spinal tracts involved in the production of HP-HSP and FP-HSP are located bilaterally in the ventrolateral quadrant and in the contralateral lateral funiculus and ventrolateral quadrant, respectively. Principal central nuclei feeding back HSP might be situated diffusely in the medulla down to the caudal part. Ketamine is suggested to suppress these inhibitory feedback potentials predominantly at, and partly even below, the level of the medulla.

Drug release was controlled by a combination of prodrug and polymer matrix. Prodrugs of 5-fluoro-2'-deoxyuridine with different physicochemical properties were synthesized by esterification with aliphatic acids (propionate, n-butyrate, and n-pentanoate). Microspheres containing these ester prodrugs were prepared with poly(3-hydroxybutyrate) of three molecular weights (65,000, 135,000, and 450,000). The release rates from the spheres depended on both the lipophilicity of the prodrug and the molecular weight of the polymer. Regardless of the polymer, the relative release rates were propionate greater than butyrate greater than pentanoate. The release of butyrate and pentanoate from the spheres consisting of low-molecular-weight polymer (M(r), 65,000) was faster than that from the spheres of higher molecular weight (M(r), 135,000 or 450,000). A single intraperitoneal injection of spheres of the highest molecular weight polymer containing butyrate or pentanoate resulted in higher antitumor effects against P388 leukemia in mice than did free prodrugs given over a period of five consecutive days. The polymer sphere itself showed low toxicity to and good biocompatibility with mice and rats.

A previous report from this laboratory showed that in vitro transcription of total genomic DNA of the newt Cynopus pyrrhogaster resulted in a discrete sized 8 S RNA, which represented highly repetitive and transcribable sequences with a glutamic acid tRNA-like structure in the newt genome. We isolated four independent clones from a newt genomic library and determined the complete sequences of three 2000 to 2400 base-pair PstI fragments spanning the 8 S RNA gene. The glutamic acid tRNA-related segment in the 8 S RNA gene contains the CCA sequence expected as the 3' terminus of a tRNA molecule. Further, the 11 nucleotides located 13 nucleotides upstream from one of the two transcription initiation sites of the 8 S RNA were found to be repeated in the region upstream from the termination site, suggesting that the original unit, which is shorter than the 8 S RNA, was retrotransposed via cDNA intermediates from the PolIII transcript. In the upstream region of the 8 S RNA gene, a 360 nucleotide unit containing the glutamic acid tRNA-related segment was found to be duplicated (clones NE1 and NE10) or triplicated (clone NE3). Except for the difference in the number of the 360 nucleotide unit, the three sequences of the 2000 to 2400 base-pair PstI fragment were essentially the same with only a few mutations and minor deletions. Inverse polymerase chain reaction and sequence determination of the products, together with a Southern hybridization experiment, demonstrated that the family consists of a tandemly repeated unit of 3300, 3700 or 4100 base-pairs. Thus during evolution, this family in the newt was created by retroposition via cDNA intermediates, followed by duplication or triplication of the 360 nucleotide unit and multiplication of the 3300 to 4100 base-pair region at the DNA level.

Poly-L-lactic acid (MW 6000) microspheres (PLA-MS) containing 5-fluoro-2'-deoxyuridine (FUdR) or four ester prodrugs of FUdR were prepared and examined with regard to the in vitro release kinetics. The incorporation efficiency of the lipophilic prodrugs into the PLA-MS was higher than that of FUdR or the hydrophilic prodrugs. The release of the lipophilic FUdR prodrugs from PLA-MS was sustained as compared with that of FUdR from PLA-MS. The release kinetics of the FUdR prodrugs appears to fit the Higuchi, Baker, and Lonsdale model in the early stage of the release process. The slope of the Baker and Lonsdale plots of the release of divaleryl-FUdR from PLA-MS decreased as the initial drug loading was decreased. The order of the release rates of FUdR prodrugs from PLA-MS with the same prodrug content was similar to that of the water solubilities of the prodrugs. These results suggest that the ester prodrugs could be released from PLA-MS by diffusion through water-filled capillaries or a series of pores rather than by diffusion through the PLA matrix.

Actin is ubiquitous in eukaryotes, nevertheless its existence has not yet been clearly proven in Tetrahymena. Here we report the cloning and sequencing of an actin gene from the genomic library of Tetrahymena pyriformis using a Dictyostelium actin gene as a probe. The Tetrahymena actin gene has no intron. The predicted actin is composed of 375 amino acids like other actins and its molecular weight is estimated as 41,906. Both T. pyriformis and T. thermophila possess a single species of actin genes which differ in their restriction patterns. Northern hybridization analysis revealed that the actin gene was actively transcribed in vivo. To detect the gene product, we synthesized an N-terminal peptide of the deduced sequence and prepared its antibody. Using an immunoblotting technique, we identified Tetrahymena actin on a two-dimensional gel electrophoretic plate. The actin spot migrated near an added spot of rabbit skeletal muscle actin, but clearly differed from the latter in its isoelectric point and apparent molecular weight. The primary structure of Tetrahymena actin shares about 75% homology equally with those of other representative actins. This value is extremely low as a homology rate between known actins. Tetrahymena actin diverges not only in relatively variable regions of other actins, but also in relatively constant regions. The hydrophilicity levels of two regions (residues 190 to 200 and residues 225 to 235) are also quite different between the Tetrahymena actin and skeletal muscle actin. Thus, we conclude that actin is present in Tetrahymena, but it is one of the most unique actins among the actins known hereto.

A simple agglutination microassay is described for studying the presence of antigenic determinants on the surface of liposomal membranes. Antigen-sensitized radioactive liposomes, reacted with specific immune IgG in the wells of a microtest plate, were passed through a glass fiber filter in a multiple cell culture harvester. The amount of agglutinate trapped on the filter as determined by the radioactivity, was proportional to the amount of antigen in the liposomes and to the amount of specific immune IgG in the reaction mixture. The specificity of the agglutination was determined by inhibition with hapten and carrier antigens. Liposomes actively sensitized with hapten-conjugated lipid, and passively sensitized with protein, were used in the assay. A new method of preparing protein-bound liposomes is also described.

To evaluate early neonatal circulatory adaptation, left and right ventricular cardiac outputs were examined in 10 normal neonates using Doppler echocardiography. Serial examinations were performed until ductal closure was confirmed. Two-dimensional echocardiography and Doppler echocardiography were used to evaluate structures of the heart and great vessels and flow within them. Then, the diameters of the ascending aorta (dAo) and pulmonary artery (dPA) were measured using M-mode echocardiography. The flow velocity patterns of the ascending aorta and pulmonary artery were recorded, measuring mean aortic (VAo) and pulmonary artery velocities (VPA). Left (LVCO) and right ventricular cardiac outputs (RVCO) were calculated as follows: LVCO = (dAo)2/4 x pi x VAo x 60, RVCO = (dPA)2/4 x pi x VPA x 60. 1. Patency of the ductus arteriosus was confirmed by the shunt through it in all neonates initially examined. The initial velocity pattern of the ductal shunts was bidirectional (9/10) or continuous left-to-right (1/10). The flow velocity pattern changed to continuous left-to-right in most neonates, and spontaneous closure of the ductus was confirmed at the age of 13 to 64.5 (mean 31.4) hrs. 2. Left-to-right shunt through the stretched foramen ovale was noted in six neonates temporarily. 3. As for the arterial diameter, dAo did not change, but dPA decreased at the time of spontaneous closure of the ductus. 4. Concerning mean aortic velocity, VAo increased when a continuous ductal left-to-right shunt was initially suspected, while VPA increased when spontaneous closure of the ductus was confirmed. 5. For cardiac output, LVCO (ml/min) changed from 618.4----718.3----562.7, while RVCO showed no change (576.1----546.5----557.8) according to a ductal flow change from bidirectional to continuous left-to-right and finally to no shunt. The LVCO/RVCO ratio increased with increasing age and (in 7/9) the maximal rate was noted when the continuous left-to-right ductal shunt was confirmed. The increased LVCO contributed to the increased LVCO/RVCO ratio. Using this method, serial evaluations of two ventricular cardiac outputs could be made.

SH-bearing subunits of IgM antibody were coupled through the Fc portion to sonicated liposomes consisting of N-(m-maleimidobenzoyl)dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylcholine and cholesterol. Liposomes coated with subunits of monoclonal IgM antibody against a mouse mammary tumor-associated antigen selectively bound to the antigenic target cells. Assays of the liposomes for complement dependent cytotoxicity and spleen cell binding revealed inactivation of the liposome-coupled antibodies in both assays.

A hot-water extract from the seed of Plantago asiatica showed a potent inhibitory activity against jack bean alpha-mannosidase, and a flavanone glucoside, plantagoside, was isolated as the inhibitor. Plantagoside was a specific inhibitor for jack bean alpha-mannosidase (IC50 at 5 microM) and appeared to be a non-competitive inhibitor of the enzyme. Whereas, negligible or weak inhibitory activities were observed for beta-mannosidase, beta-glucosidase, and sialidase tested. Plantagoside also inhibited alpha-mannosidase activities in mouse liver lysosomal and microsomal fractions, and the enzyme inhibitory activity in microsomal fraction was enhanced in the presence of glucosidase inhibitor, castanospermine. Plantagoside suppressed antibody response to sheep red blood cells and concanavalin A induced lymphocyte proliferation which was measured by [3H]thymidine incorporation.

Liposome surfaces were modified with normal rabbit IgG or rabbit anti-sheep erythrocyte IgG by adsorption or by coupling with a water soluble cross-linking reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI). Modification with normal IgG reduced the non-specific binding of the liposomes to erythrocytes. The immunological binding capacity of the immune IgG on liposomes was better for the chemically coupled preparation than for the adsorbed one. Complement dependent cytolytic activity of the immune IgG towards target erythrocytes was diminished as a consequence of liposome binding by either method, but to a lesser extent in the coupling method. Antibody coating by EDCI thus provides liposomes which can bind effectively to antigenic target cells.