Oleic Acid Activates Mitochondrial Energy Metabolism and Reduces Oxidative Stress Resistance in the Pancreatic ß-Cell Line INS-1.

Elevated concentration of saturated fatty acids in plasma adversely affects pancreatic ß-cells, but the effects of unsaturated fatty acids are controversial. In this study, we examined the effects of oleic acid (OA), a monounsaturated fatty acid, on mitochondrial function, which is important for insulin secretion, using INS-1 cells, a pancreatic ß-cell line derived from rats. Observations of mitochondrial membrane potential and intracellular ATP concentration showed that the electron transport chain was enhanced and ATP production increased in cells treated with OA, indicating that the response that occurs from sensing an increase in glucose concentration to the production of ATP was accelerated. Measurements of intracellular reactive oxygen species (ROS) indicated that the rate of increase in ROS after glucose stimulation was significantly higher in OA-treated cells. The mRNA expression levels of superoxide dismutase 1 and 2, which are responsive to ROS and other substances, were significantly increased in OA 1-d treated cells, but decreased in OA 7-d treated cells. It can be inferred that continued exposure to high concentrations of OA reduced ROS processing capacity and increased intracellular ROS levels. The mRNA expression of apoptosis-inducing enzyme Caspase-3 was significantly increased in OA-treated cells, although its activity was not high. However, the apoptosis induction rate after H2O2 stimulation was significantly higher in OA-treated cells. The high OA environment was shown to promote mitochondrial energy metabolism, leading to an increase in glucose sensitivity and a decrease in oxidative stress resistance.

Effectiveness of integrated bovine leukemia virus eradication strategies utilizing cattle carrying resistant and susceptible major histocompatibility complex class II DRB3 alleles.

Bovine leukemia virus (BLV) has spread worldwide and causes serious problems in the cattle industry owing to the lack of effective treatments and vaccines. Bovine leukemia virus is transmitted via horizontal and vertical infection, and cattle with high BLV proviral load (PVL), which is a useful index for estimating disease progression and transmission risk, are considered major infectious sources within herds. The PVL strongly correlates with highly polymorphic bovine lymphocyte antigen (BoLA)-DRB3 alleles. The BoLA-DRB3*015:01 and *012:01 alleles are known susceptibility-associated markers related to high PVL, and cattle with susceptible alleles may be at a high risk of BLV transmission via direct contact with healthy cows. In contrast, the BoLA-DRB3*009:02 and *014:01:01 alleles comprise resistant markers associated with the development of low PVL, and cattle with resistant alleles may be low-risk spreaders for BLV transmission and disrupt the BLV transmission chain. However, whether polymorphisms in BoLA-DRB3 are useful for BLV eradication in farms remains unknown. Here, we conducted a validation trial of the integrated BLV eradication strategy to prevent new infection by resistant cattle and actively eliminate susceptible cattle in addition to conventional BLV eradication strategies to maximally reduce the BLV prevalence and PVL using a total of 342 cattle at 4 stall-barn farms in Japan from 2017 to 2019. First, we placed the resistant milking cattle between the BLV-positive and BLV-negative milking cattle in a stall barn for 3 yr. Interestingly, the resistant cattle proved to be an effective biological barrier to successfully block the new BLV infections in the stall-barn system among all 4 farms. Concomitantly, we actively eliminated cattle with high PVL, especially susceptible cattle. Indeed, 39 of the 60 susceptible cattle (65%), 76 of the 140 neutral cattle (54%), and 20 of the 41 resistant cattle (48.8%) were culled on 4 farms for 3 years. Consequently, BLV prevalence and mean PVL decreased in all 4 farms. In particular, one farm achieved BLV-free status in May 2020. By decreasing the number of BLV-positive animals, the revenue-enhancing effect was estimated to be ¥5,839,262 ($39,292.39) for the 4 farms over 3 yr. Our results suggest that an integrated BLV eradication program utilization of resistant cattle as a biological barrier and the preferential elimination of susceptible cattle are useful for BLV infection control.

A novel real time PCR assay for bovine leukemia virus detection using mixed probes and degenerate primers targeting novel BLV strains.

The bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, the most common neoplastic disease in cattle. We previously developed the quantitative real-time PCR (qPCR) assay to measure the proviral loads of BLV using coordination of common motif (CoCoMo) degenerate primers. We here found four single mutations within the probe region of the original BLV-CoCoMo-qPCR assay, three of which have negative impact on its sensitivity in the probe sequences of the long terminal regions of the BLV-CoCoMo-qPCR-2 assay, using genomic DNA from 887 cows from 27 BLV-positive farms via a nationwide survey conducted in 2011 and 2017 in Japan. Therefore, the modified probes were designed to completely match the three BLV mutant strains identified here. Moreover, we examined the optimum ratio of the concentration to be mixed with the wild type and three new BLV TaqMan probes were designed here using genomic DNAs extracted from cattle naturally infected with the wild type BLV strain and three mutant strains. Finally, we successfully established an improved assay maintained the original sensitivity and reproducibility and can detect novel BLV strains.

Risk Assessment of Bovine Major Histocompatibility Complex Class II DRB3 Alleles for Perinatal Transmission of Bovine Leukemia Virus.

Perinatal transmission plays a critical role in the spread of bovine leukemia virus (BLV) infection in cattle herds. In the Holstein breed, we previously identified BLV resistant and susceptible bovine leukocyte antigen (BoLA)-DRB3 alleles, including BoLA-DRB3*009:02 and *014:01:01 with a low BLV proviral load (PVL), and *015:01 and *012:01 with a high PVL. Here, we evaluated the perinatal BLV transmission risk in dams with different BoLA-DRB3 alleles. BoLA-DRB3 alleles of 120 dam-calf pairs from five dairy farms in Japan were identified; their PVL was quantified using the BLV-Coordination of Common Motifs (CoCoMo)-qPCR-2 assay. Ninety-six dams were BLV-positive, and 29 gave birth to BLV-infected calves. Perinatal transmission frequency was 19% in dams with resistant alleles suppressed to a low PVL level, and 38% and 25% in dams with susceptible and neutral alleles that maintained high PVL levels, respectively. Notably, all calves with resistant alleles were BLV free, whereas 30% of calves with susceptible genes were infected. Thus, vertical transmission risk was extremely lower for dams and calves with resistant alleles compared to those with susceptible alleles. Our results can inform the development of effective BLV eradication programs under field conditions by providing necessary data to allow for optimal selection of dams for breeding.

Detection of helium in a fire victim: A case report.

We report here detection of helium in specimens derived from a burn autopsy case. A male was found in a burnt bedroom. Part of a heat-denatured plastic bag, sealing tape, and flexible tubing remained on his head and neck. In addition, five helium tanks were found near him. His history in conjunction with the discovery conditions suggested a suicide attempt by inhalation of helium. The body had extensive first to fourth degree burns caused by heat. A small amount of soot was deposited in the respiratory tract. Except for the thermal burns, no other injuries were found. Toxicologically, the blood carboxyhemoglobin saturation levels were less than 6%, while combustion-derived volatile hydrocarbons such as benzene or toluene were detected in the blood. In addition, tracheal gas, gastric gas, headspace gas of lung tissue, brain, and heart blood were collected during autopsy for detection of helium. Analysis was performed using headspace gas chromatography with a thermal conductivity detector. Helium was detected in all of the samples tested. Etizolam at a low limit of therapeutic concentration or less was detected in the blood. Neither ethanol nor other drugs of abuse were detected in his blood or urine. Autopsy findings and experiments suggest that the victim inhaled helium and was still alive when a fire broke out. The cause of his death was diagnosed as death from fire and flames. The present result suggests that helium may remain in a burned body and that investigation of helium in cases of fire-related deaths is informative for determination of the cause of death or confirmation of the ante mortem involvement of helium.

Changes and Variations in Death Due to Senility in Japan.

OBJECTIVE: The proportion of elderly individuals (≥65 years old) in Japan has markedly increased. However, the definition of senility in Japan is controversial. The aim of the present study was to investigate changes and variations in the number of deaths due to senility in Japan. METHODS: Information on the number of deaths due to senility between 1995 and 2018 as well as other major causes of death was obtained from the Statistics Bureau of Japan official website. Changes and variations in the number of deaths due to senility were compared with other major causes of death in Japan. The relationships between the number of deaths due to senility and socioeconomic factors were also examined in an ecological study. RESULTS: The number of deaths due to senility was 35.7 ± 23.2/one hundred thousand people/year during the observation period and has continued to increase. A change point was identified in 2004 by a Jointpoint regression analysis. Variations in the number of deaths due to senility, which were evaluated by a coefficient of variation, were significantly greater than those due to other major causes of death, i.e., malignant neoplasm, heart diseases, cerebrovascular diseases, and pneumonia. The number of elderly individuals (≥65 years old) (%) and medical bills per elderly subject (≥75 years old) correlated with the number of deaths due to senility. CONCLUSION: The number of deaths due to senility has been increasing, particularly since 2004. However, variations in the number of deaths due to senility were observed among all prefectures in Japan.

A rare maternal gastrointestinal stromal tumor found in the second trimester of pregnancy: A case report.

Gastrointestinal stromal tumors are rare, and in pregnancy they are extremely rare. We present a case of a maternal gastrointestinal stromal tumor found in the second trimester of pregnancy. A 29-year-old woman, gravida 1 para 0, complained of bloody vomiting at 14 weeks of gestation. She had no significant medical history. We performed plain computed tomography and upper gastrointestinal endoscopy. Precise examination revealed a large mass in the stomach and an exposed blood vessel on the surface. An exposed blood vessel can be harmful for mother and fetus as it might rupture during the pregnancy. We performed a distal gastrectomy at 16 weeks of gestation. Histology confirmed a localized gastrointestinal stromal tumor with a high risk of recurrence, and adjuvant imatinib was recommended. The patient elected to delay adjuvant imatinib until after delivery. The postoperative and antenatal course was favorable, and the patient was followed up by ultrasound every 2 months after the operation. After she gave birth at 40 weeks of gestation, she started adjuvant imatinib 400 mg/day. There was no evidence of recurrence 1 year after surgery. There are no guidelines for the management of gastrointestinal stromal tumors in pregnancy. Given the treatment challenges, we believe that pregnant patients should be managed by a multidisciplinary team with expertise in gastrointestinal tumors and fetal-maternal medicine.

COA-Cl induces dopamine release and tyrosine hydroxylase phosphorylation: In vivo reverse microdialysis and in vitro analysis.

We found that local perfusion of COA-Cl (0.1, 0.4, or 1.0 mM) into the dorsal striatum of living mice produced a significant and dose-dependent increase in extracellular DA levels, with the highest dose of 1.0 mM COA-Cl producing an approximately 5-fold increase in DA. Consistent with in vivo findings, 0.1 and 0.2 mM COA-Cl significantly and dose-dependently enhanced DA release 3.0 to 5.0-fold in PC12 cells, an in vitro model of DA-responsive neurons. Interestingly, the increase in striatal DA levels by COA-Cl in vivo was similar in magnitude to that observed in PC12 cells. Treatment with 0.1 mM COA-Cl significantly increased both Ser31 and Ser40 phosphorylation of tyrosine hydroxylase (TH) in PC12 cells, and Ser40 phosphorylation in iCell neurons, without altering total TH protein levels. Further, we examined whether COA-Cl could stimulate neurite outgrowth in PC12 cells and iCell neurons and found that COA-Cl significantly induced neurite outgrowth in both cell lines. Our results provide the first evidence that COA-Cl can stimulate dose-dependent DA release and activation of TH phosphorylation, suggesting that COA-Cl may be a promising therapeutic candidate for the treatment of neurological dysfunction associated with low DA.

Search and removal of radioactive seeds: another application of postmortem computed tomography prior to autopsy.

We herein introduce 3 cases illustrating a new application for pre-autopsy postmortem computed tomography (PMCT). In all 3 cases, there was insufficient background information about the victims provided to the forensic pathologists' department. PMCT showed metallic particles in the prostate gland, an indication of metallic seeds containing radioactive isotopes. In 2 of 3 cases, migrated seeds were also detected by CT imaging in the lungs and the heart. Also in 2 of 3 cases, authorities reinvestigated the victim's history before autopsy was completed, which resulted in following appropriate procedure for dealing with the seeds. Although all 3 cadavers were cremated after autopsy, the International Commission on Radiological Protection (ICRP) discourages cremation for deaths soon after radioactive seeds implantation to prevent air pollution by radioactive isotopes in the ash. Our opinion from the present cases is that pre-autopsy PMCT can be recommended for use by forensic pathologists and guidelines for investigating deaths after permanent brachytherapy should include how to deal with cadavers when medical history is limited.

Effect of terfenadine and pentamidine on the HERG channel and its intracellular trafficking: combined analysis with automated voltage clamp and confocal microscopy.

The effects of terfenadine and pentamidine on the human ether-a-go-go related gene (hERG) channel current and its intracellular trafficking were evaluated. Green fluorescent protein (GFP)-linked hERG channels were expressed in HEK293 cells, and the membrane current was measured by an automated whole cell voltage clamp system. To evaluate drug effects on channel trafficking to the cell membrane, the fraction of channel present on the cell membrane was quantified by current measurement after drug washout and confocal microscopy. Terfenadine directly blocked the hERG channel current but had no effect on trafficking of hERG channels to the cell membrane after application in culture medium for 2 d. In contrast, pentamidine had no direct effect on the hERG channel current but reduced trafficking of hERG channels. The two drugs inhibited hERG channel function through different mechanisms: terfenadine through direct channel blockade and pentamidine through inhibition of channel trafficking to the cell membrane. Combined use of automated voltage clamp and confocal microscopic analyses would provide insights into the mechanisms of drug-induced QT-prolongation and arrhythmogenesis.

Low-dose nicotine facilitates spatial memory in ApoE-knockout mice in the radial arm maze.

Here, we investigated the effects of nicotine on spatial memory in ApoE-knockout (ApoE-KO) and wild-type (WT) mice in a radial arm maze. Training occurred on three consecutive days and the test was performed on day 4, with one trial per day. Then on day 4, animals were administered nicotine (0.1, 0.25, 0.5, and 1.0 mg/kg) or the antagonist of nicotinic receptors (nAChRs) mecamylamine (MEC 2 mg/kg) alone or together with 0.1 mg/kg nicotine. The number of errors in the first eight choices was recorded. The results were that 0.1 mg/kg nicotine decreased errors in ApoE-KO mice, while 0.1 and 0.25 mg/kg nicotine reduced errors in WT mice, indicating that lower doses of nicotine elicit a memory improvement. In contrast, 1.0 mg/kg nicotine increased errors in WT mice, but not in ApoE-KO mice. MEC alone had no noticeable effect on errors in either strain of mice. However, co-administration of 0.1 mg/kg nicotine and MEC increased errors and reduced the effects of nicotine in WT mice, but not in ApoE-KO mice. Our study found a biphasic effect of nicotine in WT mice: it improves spatial memory at lower doses and impairs it at a higher dose. In ApoE-KO mice, nicotine improves memory at a low dose and has no effect at a higher dose, suggesting that the ApoE deficiency may influence the efficacy of nicotine. Moreover, a reversal of nicotinic effects with MEC was seen in WT mice, indicating the likelihood of the involvement of nAChRs in the spatial-memory response to nicotine.

Frequent somatic mosaicism of NEMO in T cells of patients with X-linked anhidrotic ectodermal dysplasia with immunodeficiency.

Somatic mosaicism has been described in several primary immunodeficiency diseases and causes modified phenotypes in affected patients. X-linked anhidrotic ectodermal dysplasia with immunodeficiency (XL-EDA-ID) is caused by hypomorphic mutations in the NF-κB essential modulator (NEMO) gene and manifests clinically in various ways. We have previously reported a case of XL-EDA-ID with somatic mosaicism caused by a duplication mutation of the NEMO gene, but the frequency of somatic mosaicism of NEMO and its clinical impact on XL-EDA-ID is not fully understood. In this study, somatic mosaicism of NEMO was evaluated in XL-EDA-ID patients in Japan. Cells expressing wild-type NEMO, most of which were derived from the T-cell lineage, were detected in 9 of 10 XL-EDA-ID patients. These data indicate that the frequency of somatic mosaicism of NEMO is high in XL-ED-ID patients and that the presence of somatic mosaicism of NEMO could have an impact on the diagnosis and treatment of XL-ED-ID patients.

Detection of base substitution-type somatic mosaicism of the NLRP3 gene with >99.9% statistical confidence by massively parallel sequencing.

Chronic infantile neurological cutaneous and articular syndrome (CINCA), also known as neonatal-onset multisystem inflammatory disease (NOMID), is a dominantly inherited systemic autoinflammatory disease and is caused by a heterozygous germline gain-of-function mutation in the NLRP3 gene. We recently found a high incidence of NLRP3 somatic mosaicism in apparently mutation-negative CINCA/NOMID patients using subcloning and subsequent capillary DNA sequencing. It is important to rapidly diagnose somatic NLRP3 mosaicism to ensure proper treatment. However, this approach requires large investments of time, cost, and labour that prevent routine genetic diagnosis of low-level somatic NLRP3 mosaicism. We developed a routine pipeline to detect even a low-level allele of NLRP3 with statistical significance using massively parallel DNA sequencing. To address the critical concern of discriminating a low-level allele from sequencing errors, we first constructed error rate maps of 14 polymerase chain reaction products covering the entire coding NLRP3 exons on a Roche 454 GS-FLX sequencer from 50 control samples without mosaicism. Based on these results, we formulated a statistical confidence value for each sequence variation in each strand to discriminate sequencing errors from real genetic variation even in a low-level allele, and thereby detected base substitutions at an allele frequency as low as 1% with 99.9% or higher confidence.

High incidence of NLRP3 somatic mosaicism in patients with chronic infantile neurologic, cutaneous, articular syndrome: results of an International Multicenter Collaborative Study.

OBJECTIVE: Chronic infantile neurologic, cutaneous, articular (CINCA) syndrome, also known as neonatal-onset multisystem inflammatory disease (NOMID), is a dominantly inherited systemic autoinflammatory disease. Although heterozygous germline gain-of-function NLRP3 mutations are a known cause of this disease, conventional genetic analyses fail to detect disease-causing mutations in ∼40% of patients. Since somatic NLRP3 mosaicism has been detected in several mutation-negative NOMID/CINCA syndrome patients, we undertook this study to determine the precise contribution of somatic NLRP3 mosaicism to the etiology of NOMID/CINCA syndrome. METHODS: An international case-control study was performed to detect somatic NLRP3 mosaicism in NOMID/CINCA syndrome patients who had shown no mutation during conventional sequencing. Subcloning and sequencing of NLRP3 was performed in these mutation-negative NOMID/CINCA syndrome patients and their healthy relatives. Clinical features were analyzed to identify potential genotype-phenotype associations. RESULTS: Somatic NLRP3 mosaicism was identified in 18 of the 26 patients (69.2%). Estimates of the level of mosaicism ranged from 4.2% to 35.8% (mean ± SD 12.1 ± 7.9%). Mosaicism was not detected in any of the 19 healthy relatives (18 of 26 patients versus 0 of 19 relatives; P < 0.0001). In vitro functional assays indicated that the detected somatic NLRP3 mutations had disease-causing functional effects. No differences in NLRP3 mosaicism were detected between different cell lineages. Among nondescript clinical features, a lower incidence of mental retardation was noted in patients with somatic mosaicism. Genotype-matched comparison confirmed that patients with somatic NLRP3 mosaicism presented with milder neurologic symptoms. CONCLUSION: Somatic NLRP3 mutations were identified in 69.2% of patients with mutation-negative NOMID/CINCA syndrome. This indicates that somatic NLRP3 mosaicism is a major cause of NOMID/CINCA syndrome.

Rapid diagnosis of FHL3 by flow cytometric detection of intraplatelet Munc13-4 protein.

Familial hemophagocytic lymphohistiocytosis (FHL) is a potentially lethal genetic disorder of immune dysregulation that requires prompt and accurate diagnosis to initiate life-saving immunosuppressive therapy and to prepare for hematopoietic stem cell transplantation. In the present study, 85 patients with hemophagocytic lymphohistiocytosis were screened for FHL3 by Western blotting using platelets and by natural killer cell lysosomal exocytosis assay. Six of these patients were diagnosed with FHL3. In the acute disease phase requiring platelet transfusion, it was difficult to diagnose FHL3 by Western blot analysis or by lysosomal exocytosis assay. In contrast, the newly established flow cytometric analysis of intraplatelet Munc13-4 protein expression revealed bimodal populations of normal and Munc13-4-deficient platelets. These findings indicate that flow cytometric detection of intraplatelet Munc13-4 protein is a sensitive and reliable method to rapidly screen for FHL3 with a very small amount of whole blood, even in the acute phase of the disease.

Relative contributions of 5-hydroxytryptamine (5-HT) receptor subtypes in 5-HT-induced vasoconstriction of the distended human saphenous vein as a coronary artery bypass graft.

It is established that the segment of saphenous vein (SV) that is widely used as a conduit vessel in coronary artery bypass graft (CABG) surgery is distended with high pressure to check for leaks and to increase the patency before implantation into coronary arterial circulation. The aim of the present study was to elucidate the relative contributions of 5-hydroxytryptamine (5-HT) receptor subtypes responsible for 5-HT-induced vasoconstriction of the distended human SV. Whereas about half of the 5-HT-induced vasoconstriction still remained in the presence of supramaximum concentration of sarpogrelate or of SB224289 (5-HT(2A) and 5-HT(1B) receptor antagonists, respectively), simultaneous treatment with sarpogrelate and SB224289 almost completely inhibited the 5-HT-induced vasoconstriction. Immunopositive staining for 5-HT(2A) and 5-HT(1B) receptors was detected in smooth muscle cells of the distended human SV and there was no significant difference between the immunopositive areas of 5-HT(2A) and 5-HT(1B) receptors. These results demonstrate that 5-HT(2A) and 5-HT(1B) receptors similarly contribute to 5-HT-induced vasoconstriction in human distended SV. Thus, when the SV is used as a CABG conduit, a combination of 5-HT(2A) and 5-HT(1B) receptor antagonists would appear to be most useful to prevent 5-HT-induced spasm.

Effects of systemic nicotine, alcohol or their combination on cholinergic markers in the frontal cortex and hippocampus of rat.

Acute alcohol (Alc) intoxication has been shown to decrease choline acetyltransferase (ChAT) in the rat brain. The present study extends that finding by examining the effects of nicotine (Nic), Alc, and their combination on ChAT and acetylcholinesterase (AChE) in the frontal cortex and hippocampus of rat. The samples were collected at 30 and 120 min after intraperitoneal administration of saline (0.9%, control), Nic (1 mg/kg), Alc (1 g/kg), and Nic + Alc and analyzed by RT-PCR, Western blot and colorimetry. Alc alone considerably reduced ChAT mRNA expression, whereas Nic alone decreased AChE mRNA expression. In contrast, Nic + Alc exposure had resulted in no significant change in the parameters. These findings are consistent with the results of the Western blot and AChE activity analysis. The results, therefore, indicate that Nic and Alc alone may interact with the central cholinergic system. This interactive effect may contribute to a frequent association of tobacco and Alc consumption.

An autopsy case of butane gas abuse.

A case of fatal butane gas poisoning in a young female is presented. Quantitative toxicological analysis showed that the concentration of butane in the femoral blood was 6.8 microl/ml, and isobutane and propane were also identified. Severe congestion of the lungs and deposition of lipofuscin in the myocardium were also observed. We concluded that the cause of death of the victim was due to cardiac arrhythmia induced by the butane gas abuse.

[Pharmacological studies on responsiveness of 5-hydroxytryptamine in overcoming perioperative spasm of coronary artery bypass graft].

In ischemic heart diseases, the use of the internal thoracic artery (ITA) as an arterial graft has been associated with longer survival and better quality of life. However, it has been reported that vasospasm of the ITA graft frequently occurs and increases perioperative and postoperative morbidity. Serotonin (5-HT) plays an important role in the occurrence of vasospasm. We examined 5-HT receptor subtypes responsible for the 5-HT-induced vasocontraction in the human ITA. The contractile response caused by 5-HT was mediated by activation of not only 5-HT(2A) receptors but also 5-HT(1B) receptors. We also examined the relationship between 5-HT-induced vasocontraction of the rabbit femoral artery and arteriosclerosis using the arteriosclerosis model of repeated balloon-injury. The contractile response caused by 5-HT in the femoral artery with arteriosclerosis was significantly greater than that in the normal artery. Additionally, we demonstrated that insulin induced internalization of 5-HT(2A) receptors from the plasma membrane in HEK293 cells. Diabetes mellitus (DM) is a risk factor for ischemic heart diseases. We evaluated the 5-HT-induced vasocontraction, mediated by activation of 5-HT(2A) and 5-HT(1B) receptors, in the ITA obtained from patients with DM or without DM undergoing coronary bypass surgery. The contractile response caused by 5-HT in the ITA from patients with DM was significantly greater than that from patients without DM. Our findings suggest that when the ITA is used as an arterial graft, simultaneous treatment with 5-HT(2A) and 5-HT(1B) receptor antagonists is useful to prevent 5-HT-induced vasospasm, especially in patients with DM.

Efficient in vivo gene transfection by stable DNA/PEI complexes coated by hyaluronic acid.

Plasmid DNA was mixed with polyethyleneimine (PEI) and hyaluronic acid (HA) to afford ternary complexes with negative surface charge regardless of the mixing order. They showed reduced non-specific interactions with blood components. When DNA and PEI were mixed at a high concentration such as that used in in vivo experiments, they soon aggregated, and large particles were formed. On the other hand, pre-addition of HA to DNA prior to PEI effectively diminished the aggregation, and 10% (in volume) of the complexes remained as small particles with a diameter below 80 nm. Those negatively charged small ternary complexes induced a much stronger extra-gene expression in tumor than binary DNA/PEI complex after intratumoral or intravenous injection into the mice bearing B16 cells.