TB-related technical enquiries received in Japan, 2017-2019.

SETTING: Japan, an intermediate TB burden country. OBJECTIVE: To review TB-related technical enquiries received at the Research Institute of Tuberculosis, Japan, from January 2017 to December 2019. DESIGN: This was a cohort study. RESULTS: A total of 2,197 enquiries were analysed. On average, 61.0 enquiries/month (range: 42-81) were received. The enquiry rates were highest for the Yamanashi (4.65/100,000 population) and Ishikawa (4.55) Prefectures, and lowest in the Yamagata (0.46) and Tochigi (0.56) Prefectures. The main organisations the enquirers belonged to were local governments (n = 1,585, 72.1%) and healthcare facilities (n = 307, 14.0%). The enquirers were medical doctors (n = 391, 17.8%), nurses (n = 1,207, 54.9%), other healthcare professionals (n = 57, 2.6%), the general public (n = 168, 7.6%) and others/unknown (n = 374, 17.0%). The most frequent enquiries were about TB diagnosis and treatment (n = 501, 22.8%), including laboratory diagnosis (n = 88, 4.0%), TB treatment in general (n = 93, 4.2%) and management of comorbidities (n = 86, 3.9%), followed by contact investigations (n = 385, 17.5%) and TB in foreigners (n = 344, 15.7%). CONCLUSION: As the most frequent enquiries were about diagnosis and treatment of TB, the health ministry of Japan should maintain a few specialised TB institutions with TB physicians to provide technical assistance.

LIEU: Le Japon, un pays à charge intermédiaire en matière de TB. OBJECTIF: Examiner les demandes de renseignements techniques liées à la TB reçues au Research Institute of Tuberculosis, au Japon, de janvier 2017 à décembre 2019. METHODE: Il s'agissait d'une étude de cohorte. RÉSULTATS: Au total, 2 197 demandes ont été analysées. En moyenne, 61,0 demandes de renseignements/mois (fourchette : 42­81) ont été reçues. Les taux de demande étaient les plus élevés dans les préfectures de Yamanashi (4,65/100 000 habitants) et d'Ishikawa (4,55), et les plus faibles dans les préfectures de Yamagata (0,46) et de Tochigi (0,56). Les principales organisations auxquelles appartiennent les enquêteurs sont les administrations locales (n = 1 585 ; 72,1%) et les établissements de santé (n = 307; 14,0%). Les enquêteurs étaient des médecins (n = 391 ; 17,8%), des infirmières (n = 1 207 ; 54,9%), d'autres professionnels de la santé (n = 57 ; 2,6%), le grand public (n = 168 ; 7,6%) et autres/inconnus (n = 374 ; 17,0%). Les demandes les plus fréquentes concernaient le diagnostic et le traitement de la TB (n = 501 ; 22,8%), y compris le diagnostic en laboratoire (n = 88 ; 4,0%), le traitement de la TB en général (n = 93 ; 4,2%) et la prise en charge des comorbidités (n = 86 ; 3,9%), suivis par les enquêtes sur les contacts (n = 385 ; 17,5%) et la TB chez les étrangers (n = 344 ; 15,7%). CONCLUSION: Comme les demandes de renseignements les plus fréquentes concernaient le diagnostic et le traitement de la TB, le ministère de la santé du Japon devrait maintenir quelques institutions spécialisées dans la TB avec des médecins spécialistes de la TB pour fournir une assistance technique.

Glutathione ethyl ester improved the age-induced decline in the developmental competence of bovine oocytes.

The aberrant redox regulation and anti-oxidative defense is one of the main causes of age-induced decline in oocytes quality and embryo development in mammals. The present study aimed to elucidate the effect of glutathione ethyl ester (GSH-OEt), a cell-permeable glutathione (GSH) donor, on the developmental competence of oocytes in cows with advanced reproductive age. Oocytes were collected from cows aged 30-50 months or >120 months, which were defined as young or aged, respectively, and subjected to in vitro maturation (IVM) in the presence of 5 mM of GSH-OEt. In aged cows, the GSH level in follicular fluid was lower, and the intracellular levels of reactive oxygen species (ROS) in post-IVM oocytes was higher than those in young cows. GSH-OEt supplementation during IVM reduced the ROS contents of oocyte in aged cows but not in young cows. GSH-OEt treatment promoted the meiotic progression and increased the proportion of oocytes with mature cytoplasm containing evenly dispersed cortical granules in aged cows. After in vitro fertilization, the normal fertilization and development to the blastocyst stage were enhanced by GSH-OEt in aged cows to levels comparable to those in young cows. Further, oocyte maturation in the presence of GSH-OEt increased the proportion of diploid blastocyst in aged cows. In contrast, GSH-OEt failed to enhance the oocyte maturation, fertilization, and embryo development in young cows. Taken together, the exogenous supplementation of GSH-OEt during IVM modulated the age-related oxidative damage of bovine oocytes and improved the developmental competence of oocytes in aged cows. Oocytes presented a distinct response to GSH-OEt treatment depending on the donor age. GSH-OEt supplementation during IVM could be of practical value through the efficiency improvement of chromosomally normal embryo production in aged cows.

Developmental kinetics and viability of bovine embryos produced in vitro with sex-sorted semen.

The present study aimed to elucidate the developmental kinetics, growth potential, and viability of bovine embryos produced in vitro with sexed semen. Bovine oocytes were fertilized in vitro using unsorted and X-sorted semen from the same Holstein bulls, and the kinetics of in vitro development were continuously monitored for 10 d through time-lapse cinematography. The blastocyst formation rate was lower in the X-sorted group than in the unsorted group (P < 0.01), whereas the normal fertilization rate did not differ between groups. Morphokinetic evaluation revealed that the incidence of blastomere fusion during the first cleavage division, termed reverse cleavage, was higher in the X-sorted group (P < 0.01). Furthermore, embryos produced with X-sorted semen showed slower growth throughout the developmental period than embryos produced with unsorted semen (P < 0.01). The cell number of the trophectoderm and inner cell mass of blastocysts was reduced in the X-sorted group (P < 0.01). In embryos that developed to the blastocyst stage, the hatchability (P < 0.05), chromosomal normality (P < 0.01), and survivability after the conventional frozen-thawing process (P < 0.05) were reduced in the X-sorted group compared to that in the unsorted group, indicating a compromised viability of embryos derived from X-sorted semen. Taken together, the first cleavage dysmorphism, delayed embryo growth, and impaired viability of embryos developed to the blastocyst stage may explain the mechanism of reduced fertility in embryos derived from sexed semen. The kinetic evaluation of early embryo development and de-selection of embryos presenting the aberrant first cleavage would be valid for clinical application to produce sexed embryos with high implantation potential.

Growth potential of bovine embryos presenting abnormal cleavage observed through time lapse cinematography.

Time-lapse monitoring (TLM) has emerged as a novel technology for the continuous and noninvasive evaluation of embryos. TLM has revealed the prevalence of specific dysmorphisms such as abnormal development during the early-cleavage stage of embryos. However, little information is available on the prevalence and consequences of abnormal cleavage in bovine embryos. Hence, this study aimed to investigate growth potential of bovine embryos presenting abnormal cleavage, such as reverse cleavage (RC), direct cleavage (DC), and irregular and unsmooth ruffling of the oolema membrane (ruffling). Bovine embryos derived through in vitro fertilization (IVF) were cultured in the microwell culture dishes, and the kinetics of in vitro development were observed through TLM at 20-min intervals for 10 d. Approximately 36% of embryos that developed into a blastocyst presented abnormal cleavage. Morphokinetic evaluations revealed that RC, DC, and ruffling embryos showed slower development compared to embryos with normal cleavage (P < 0.01). Embryos with RC and DC, but not ruffling, revealed impaired hatchability (P < 0.05) with increased collapses of the blastocyst cavity until hatching (P < 0.0001). Moreover, the RC and DC embryos presented increased chromosomal aneuploidy (P < 0.05). These results suggest a compromised viability of embryos with RC and DC. This is the first report that clarified the effect of abnormal cleavage on the morphokinetics and growth potential of bovine IVF embryos. Results indicate that the kinetic evaluation of bovine embryos using the time-lapse imaging system will be beneficial for selecting embryos with a high viability.

Tuberculosis-related technical enquiries received by a national level institution in Japan, 2014-2016.

Background: Japan had a tuberculosis (TB) notification rate of 13.9 per 100 000 population in 2016. Objectives: To characterise TB-related enquiries received by the Research Institute of Tuberculosis, Tokyo, Japan, between January 2014 and December 2016. Design: A descriptive study of the time, place and other attributes of the enquiries. Results: A total of 1864 enquiries were listed for analysis. On average, 51.8 enquiries (range 30-77) were received per month. The enquiry rates were highest for Yamanashi (5.87/100 000) and Kochi (5.77) Prefectures, and lowest in Miyazaki (0.45) and Saga (0.48) Prefectures. Enquirers belonged mostly to local governments (n = 1212, 65%) and health care facilities (n = 386, 21%), and included medical doctors (n = 412, 22%), nurses (n = 926, 50%) and the general public (n = 150, 8.0%). Most enquiries concerned TB diagnosis and treatment (n = 475, 25%), including diagnosis in general (n = 38, 2.0%), laboratory diagnosis (n = 83, 4.5%), anti-tuberculosis treatment in general (n = 62, 3.3%) and the management of comorbidities or adverse events (n = 60, 3.2%), followed by contact investigations (n = 371, 20%). Conclusions: As most enquiries concerned the diagnosis and treatment of TB, the Ministry of Health of Japan should maintain a number of specialised TB institutions with TB clinicians to provide technical assistance.

Contexte : Le Japon avait un taux de notification de la tuberculose (TB) de 13,9 par 100 000 habitants en 2016.Objectif : Caractériser les demandes liées à la TB reçues par le Research Institute of Tuberculosis, Tokyo, Japon, de janvier 2014 à décembre 2016.Schéma : Une étude descriptive en termes de date, lieu et autres caractéristiques des demandes.Résultats : Un total de 1864 demandes ont été listées pour l'analyse. En moyenne, 51,8 demandes (fourchette 30­77) par mois ont été reçues. Les taux de demande ont été les plus élevés pour les préfectures de Yamanashi (5,87/100 000 habitants) et de Kochi (5,77) et les plus bas dans les préfectures de Miyazaki (0,45) et de Saga (0,48). Les organisations principales auxquelles appartenaient les demandeurs ont été les gouvernements locaux (n = 1212 ; 65%) et les structures de soins de santé (n = 386 ; 21%). Les demandeurs ont été des médecins (n = 412 22%), des infirmiers (n = 926 ; 50%) et le public général (n = 150 ; 8,0%). Les demandes les plus fréquentes ont été liées au diagnostic et au traitement de la TB (n = 475 ; 25%), incluant le diagnostic en général (n = 38 ; 2.0%), le diagnostic de laboratoire (n = 83 ; 4,5%), le traitement de la TB en général (n = 62 ; 3,3%) et la prise en charge des comorbidités ou des effets secondaires (n = 60 ; 3,2%), suivies par la recherche des contacts (n = 371 ; 20%).Conclusion : Comme les demandes les plus fréquentes ont été relatives au diagnostic et au traitement de la TB, le Ministère de la santé du Japon devrait maintenir quelques institutions spécialisées dans la TB avec des cliniciens qui pourraient fournir une assistance technique.

Marco de referencia: En el Japón, la tasa de notificación de tuberculosis (TB) fue 13,9 por 100 000 habitantes en el 2016.Objetivos: Caracterizar las consultas relacionadas con la TB que se recibieron en el Instituto de Investigación en Tuberculosis, Tokyo, Japón, de enero del 2014 a finales de diciembre del 2016.Método: Se llevó a cabo un estudio descriptivo de las consultas con respecto al tiempo, el lugar y otros atributos de las mismas.Resultados: Se registraron 1864 consultas para análisis. En promedio, se recibieron 51,8 consultas por mes (entre 30 y 77). La tasa de consultas más alta correspondió a las prefecturas de Yamanashi (5,87/100 000 habitantes) y Kochi (5,77) y la más baja a las prefecturas de Miyazaki (0,45) y Saga (0,48). Las principales organizaciones de los solicitantes pertenecían a los gobiernos locales (n = 1212; 65%) y a los establecimientos de salud (n = 386; 21%). Los solicitantes fueron médicos (n = 412; 22%), enfermeros (n = 926; 50%) y el público en general (n = 150; 8,0%). Las consultas más frecuentes se relacionaban con el diagnóstico y el tratamiento de la TB (n = 475; 25%), entre otros, el diagnóstico en general (n = 38; 2,0%), el diagnóstico en el laboratorio (n = 83; 4,5%), el tratamiento antituberculoso en general (n = 62; 3,3%) y el manejo de las enfermedades intercurrentes o las reacciones adversas (n = 60; 3,2%), seguidos de la investigación de contactos (n = 371; 20%).Conclusiones: Dado que las consultas más frecuentes se referían al diagnóstico y el tratamiento de la TB, el Ministerio de Salud del Japón debe conservar algunas instituciones especializadas en TB con médicos experimentados que presten asistencia técnica en esta esfera.

Evaluation of tuberculosis contact investigations in Japan.

SETTING: Although monitoring and evaluation using standardised indicators is an important aspect of tuberculosis (TB) contact investigation, no attempts have been made to systematically evaluate contact investigations in Japan. OBJECTIVE: To evaluate TB contact investigations conducted in public health centres (PHCs) by estimating the scores of selected indicators. DESIGN: A cross-sectional study was conducted in 2012 to estimate six selected indicator scores for contact tracing, contact evaluation and contact treatment at 11 PHCs using the TB registry and relevant contact investigation records of all forms of newly notified active TB cases. Indicator scores were compared across PHCs using median and interquartile range (IQR). RESULTS: A total of 2527 contacts of 313 index TB cases were identified; of these, 1874 were evaluated using the tuberculin skin test (TST) or interferon-gamma release assays (IGRAs). Of 187 contacts who were TST/IGRA-positive, 15 were diagnosed with active TB. Consistently higher medians and lower IQRs were obtained for indicators of contact tracing and contact evaluation than those of contact treatment. CONCLUSION: Our study is the first to evaluate the performance of TB contact investigations in Japan using standardised indicators; the study indicated performance gaps, especially in the treatment for latent tuberculous infection among contacts.

Can social network analysis assist in the prioritisation of contacts in a tuberculosis contact investigation?

OBJECTIVES: To evaluate the effectiveness of social network analysis (SNA) in prioritising contacts in a tuberculosis (TB) contact investigation. METHOD: We reviewed and analysed patient and contact investigation data from a large outbreak that occurred in Tokyo, Japan, between 2010 and 2012. Relevant data were extracted to create a social matrix, which was then analysed using SNA software to visualise the network and calculate SNA metrics (degree and betweenness) for all patients and contacts. Statistical analyses were conducted to examine whether degree and betweenness centrality scores could prioritise contacts for in-depth investigation by calculating the odds of latent tuberculous infection (LTBI) being diagnosed among contacts with high scores compared to those with low scores. RESULTS: The data on a total of 8 patients and 376 contacts, of whom 56 were diagnosed with LTBI, were analysed. Centrality scores did not show a statistically significant association with the risk of contacts being diagnosed with LTBI. However, contacts with high betweenness scores were more likely to be diagnosed with LTBI than contacts with lower scores (OR 2.88, 95%CI 1.31-5.83, P = 0.007). CONCLUSION: Our results showed the potential of a betweenness score in prioritising contacts during TB contact investigation.

Myostatin alters glucose transporter-4 (GLUT4) expression in bovine skeletal muscles and myoblasts isolated from double-muscled (DM) and normal-muscled (NM) Japanese shorthorn cattle.

The purpose of this study was to determine whether myostatin alters glucose transporter-4 (GLUT4) expression in bovine skeletal muscles and myoblasts isolated from double-muscled (DM) and normal-muscled (NM) Japanese Shorthorn cattle. Plasma concentrations of glucose were lower in DM cattle than in NM cattle (P < 0.01). The expression of GLUT4 messenger RNA (mRNA) in the skeletal muscle ex vivo and in myoblasts at 72 h after differentiation in vitro was higher in DM cattle than in NM cattle (P < 0.01). In contrast, the NM and DM cattle did not differ with respect to skeletal muscle expression of GLUT1 and myocyte enhancer factor-2c (MEF2c), a transcription factor of GLUT4. In differentiated myoblasts, the expression of GLUT1, GLUT4, and MEF2c mRNAs was greater in DM cattle than in NM cattle (P < 0.01). In the presence and absence of insulin, glucose uptake in myoblasts was increased in DM cattle relative to that of NM cattle (P < 0.01). The addition of myostatin decreased the expression of GLUT4 and MEF2c mRNAs in DM myoblasts (P < 0.05). Results of the present study suggest that myostatin inhibits the expression of GLUT4 mRNA possibly via MEF2c and that the greater ability of the DM cattle to produce muscle relative to the NM cattle may be due to their greater sensitivity to insulin and greater use of glucose.

[A case of urinary retention secondary to aseptic meningitis].

A case of a 37-year-old man with urinary retention secondary to aseptic meningitis is described. He was admitted to our hospital with complaints of fever, nausea, headache, and gait disturbance. He had begun treatment 1 week previously for high fever, headache and joint pain at an outpatient department. On admission, neurological examination revealed neck stiffness, spasticity and hyperreflexia of the extremities, hypesthesia of the lower extremities, and ataxic gait. A spinal fluid examination revealed aseptic meningitis. Disturbed consciousness, diplopia, aphasia, tetraparesis, and urinary retention appeared after admission. Computed tomography and magnetic resonance imaging (MRI) of the head revealed no abnormal lesions. On the 2nd day of hospitalization, a Foley catheter was inserted for urinary retention. The patient was treated with conservative therapy, and his symptoms gradually resolved. The Foley catheter was removed on hospital day 13, but bladder dysfunction was persisted. MRI of the lumbar spine revealed herniated lumbar discs at level L3-4, L4-5, and L5-S1, but the nerve roots were not compressed. The patient was managed with clean intermittent catheterization for more than two weeks. Cystometry revealed atonic bladder. Nineteen cases of urinary retention secondary to aseptic meningitis have been reported in the previous literature. We reviewed their clinical presentation and treatment.

[Regional cerebral blood flow values measured by Xe-CT during the attack of transient global amnesia: a case report].

A 50-year-old right-handed woman was referred to our hospital for further examination of sudden global amnesia. The patient had no history of epilepsy, head injury or cerebral vascular diseases. There were no neurological deficits except for recent memory disturbance. We examined her cerebral blood flow(CBF) immediately during transient global amnesia(TGA) by stable xenon enhanced CT scans(Xe-CT) and twice thereafter. Xe-CT during the attack showed a significant diminution of regional blood flow from the right posterior temporal lobe to the occipital lobe. Conventional MRI scans also had been performed serially but it could not detect the local ischemic event. The ischemic lesion was clearly divided and larger than those cases reported using PET, SPECT, and diffusion-weighted MRI. TGA happens suddenly, and recovery is immediate. It becomes very difficult to study CBF during TGA attack. However stable Xe-CT is capable of examining CBF easily, so we concluded that CBF examination by Xe-CT in TGA patients would be helpful to reveal the mechanism of TGA.

Childhood subarachnoid hemorrhage associated with fibromuscular dysplasia.

A rare case of childhood subarachnoid hemorrhage (SAH) is reported. A 5-year-old boy presented with SAH. A cerebral angiogram showed obstruction of the first segment of the left anterior cerebral artery (A1). The A1 was surgically trapped to prevent rebleeding. Proliferation of myofibroblasts was revealed in the intima of the A1. Cerebral infarction appeared in the territory of the left middle cerebral artery (MCA) on day 2 after onset of SAH, and the patient suffered a second SAH on day 26. The second angiogram showed stenosis and a saccular aneurysm in the left MCA. A second surgery, neck clipping of the aneurysm, was needed. The multiple arterial lesions and histological findings indicated that the SAHs and the infarction were caused by the dissecting aneurysm affected with fibromuscular dysplasia (FMD). Intracranial FMD is mainly detected in children and causes cerebral ischemia and bleeding. Surgical treatment of childhood SAH should be discretely chosen with due consideration for FMD.

Nuclear transfer of blastomeres expressing EGFP-reporter gene may improve the efficiency of transgenic cattle.

The effect of timing of microinjection of DNA constructs on the efficiency of transgenic embryo production and improved efficiency and quality through combining EGFP as a reporter gene with nuclear transfer techniques were examined. From 12 to 24 h after insemination, constructs of pCXNeo-EGFP were microinjected into a pronucleus of bovine IVM-IVF zygotes. Due to the difficulty in visualizing pronuclei, the incidence of successful injection of linear DNA was higher when zygotes were injected between 20 and 24 h, as compared with an early period between 12 and 16 h after insemination. However, developmental competence of DNA-injected zygotes and the EGFP expression rate were not affected by the injection time. A majority of the embryos expressing EGFP signal were mosaic. Following nuclear transfer of blastomeres expressing EGFP, 4.5% of morulae that developed from the NT eggs had a strong EGFP signal in all live blastomeres. In other embryos, EGFP signal had been lost. When cells derived from the EGFP-positive NT morulae were subcultured, all the cells expressed strong EGFP signal at the second passage and demonstrated neomycin resistance. These results show that transient expression of nonintegrated EGFP appears frequently in EGFP-positive bovine embryos and that additional selection of EGFP-positive morulae after nuclear transfer of EGFP-positive blastomeres would facilitate selection of transgenic embryos.

[Two cases of elderly patients with ruptured AVM with contrast medium extravasation during cerebral angiography].

Two cases are reported of elderly patients who experienced intracranial extravasation of contrast medium (CM) during carotid angiography (CAG) for ruptured cerebral arteriovenous malformations (AVM). The first patient, an 87-year-old male with no history of hypertension, was admitted immediately following a loss of consciousness after swimming in a pool. CT scan revealed a large intracranial hematoma in the left frontal lobe. CAG performed 1 hour after his arrival revealed a small AVM, fed by the left anterior cerebral artery with concomitant extravasation of CM. The patient's condition subsequently deteriorated and he died the following day. The second patient, a 71-year-old female, was admitted to our hospital in a comatose state after complaining of a severe headache. CT scan revealed a right parietal lobe hemorrhage extending into the ventricles. CAG was performed and demonstrated a small AVM in the right parietal lobe with extravasation of CM. Following emergency removal of the hematoma and AVM, the patient regained consciousness although some motor deficits persisted. A literature review revealed that only 6 cases of CM extravasation with ruptured AVM have been previously reported. The 4 previous cases involved patients 9, 15, 33 and 66-year-old, the younger three of which had a good outcome. The patients reported here were much older, and had a much less favorable outcome. Thus, AVM with CM extravasation may have a better prognosis in younger individuals.

Gene transfer into hepatoma cells mediated by galactose-modified alpha-helical peptides.

To develop a receptor-mediated gene delivery system into hepatoma cells using the cationic alpha-helical peptide as the gene carrier molecule, we modified an alpha-helical peptide, which is known to have transfection abilities into cells, with a multi-antennary ligand containing several galactose residues that provide efficient binding to the asialoglycoprotein receptor. The galactose-modified peptides formed complexes with a plasmid DNA and showed gene transfer abilities into HuH-7 cells, a human hepatoma cell line. The transfection efficiency of the peptide was increased by increasing the number of modified galactose residues on the peptide. Furthermore, considerable inhibition of the transfection efficiency by the addition of asialofetuin, which is a ligand for the asialoglycoprotein receptor, was observed in all galactose-modified peptides. Based on this result, we could confirm that the internalization of the galactose-modified peptides occurred by the receptor-mediated endocytosis pathway. In addition, to understand the transport route of the peptide-DNA complex in the cell, the effects on the transfection efficiencies with several endocytosis inhibitors were examined. As a result, it was suggested that the translocation of the peptide-DNA complex from the endocytic compartments to the cytosol mainly occurred during an early endosome step.

Influence of lipophilic groups in cationic alpha-helical peptides on their abilities to bind with DNA and deliver genes into cells.

For the purpose of achieving gene transfer into cells mediated by peptides with a short chain length, we employed two kinds of amphiphilic alpha-helix peptides, mastoparan (INLK-ALAA-LAKK-IL-NH2) obtained from wasp venom and an alpha-helix model peptide (LARL-LARL-LARL-NH2). Furthermore, to strengthen the hydrophobicity of the peptide required for the formation of the aggregates with the DNA, we modified these peptides using several lipophilic groups, i.e. acyl groups with a single chain, a dialkylcarbamoyl group and a cholesteryloxycarbonyl group. We examined the ability of the peptides and their derivatives to bind and aggregate with plasmid DNA, the structural change in the peptides caused by binding with the DNA and the in vitro gene transfer abilities into COS-7 cells. As a result, mastoparan was found to acquire the DNA binding ability by introduction of the lipophilic group. The conformational change in the peptides depended on the hydrophobicity of the introduced acyl group. The DNA complex of most lipophilic mastoparan derivatives could be incorporated into the cells via the endocytosis pathway. In the case of the helix model peptide, the acyl group with a moderate chain length was required for the formation of the aggregate which is competent for incorporation into the cells. In this study, we succeeded in giving such short peptides sufficient gene transfer ability by modifying them with some lipophilic groups. However, the influence of the modification by the lipophilic groups on the formation of aggregates with DNA and the gene transfer ability depended on the structure of the peptide portion. These results indicate that consideration of total hydrophobicity balance is needed for the design of an efficient gene carrier peptide.

Chain length of cationic alpha-helical peptide sufficient for gene delivery into cells.

To define the minimal peptide length needed for gene delivery into mammalian cells, we synthesized several peptides with shortened chain lengths from the amino-termini of the original amphiphilic peptides (4(6), Ac-LARL-LARL-LARL-LRAL-LRAL-LRAL-NH( 2,) and Hel 11-7, KLLK-LLLK-LWKK-LLKL-LK), which have been known to have gene transfer abilities into cells. Each synthetic peptide was studied for its ability to bind and aggregate with plasmid DNA and the structural change of the peptide caused by binding with the DNA to establish a relative in vitro gene transfection efficiency in COS-7 cells. As a result, the deletion of eight amino acid residues of 4(6) had little influence on their ability, whereas that of 12 amino acid residues remarkably reduced the abilities to make aggregates and transfer the DNA into the cell. In the case of the Hel 11-7 series peptides, deletion of amino acid residues caused a considerable reduction in abilities to bind and form aggregates with DNA and to transfer the DNA into cell in due order. In summary, 16 and 17 amino acid residues were sufficient to form aggregates with the DNA and transfer the DNA into the cells in the deletion series of 4(6) and Hel 11-7, respectively. Furthermore, it was indicated that reduction of membrane perturbation activity of the peptide-DNA complex due to deletion of the peptide chain length caused suppression of the transfection efficiency even if the complex was incorporated into the cells. Transfer of the complex to cytosol mediated by membrane perturbation activity of the peptide is an important step for efficient protein expression from its cDNA. The results of this study will make it easy to design and synthesize a functional gene carrier molecule such as a carbohydrate-modified peptide used in targeted gene delivery.

Adsorption characteristics of tryptic fragments of bovine beta-lactoglobulin on a stainless steel surface.

As a strategy for the analysis of the mode of protein adsorption onto stainless steel surfaces, peptides obtained by tryptic digestion of bovine beta-lactoglobulin were subjected to adsorption experiments after identification of their primary structures. In the presence of 1 mM KOH, the peptides were scarcely adsorbed onto the surfaces of stainless steel particles from the peptide mixture. The adsorption experiments on isolated peptides showed that the affinities of the peptides for stainless steel surfaces in the presence of 1 mM HNO3 were significantly different from each other. Peptides without any acidic amino acid residues were scarcely adsorbed onto the surface, whereas some peptides with acidic amino acid residues were found to be irreversibly adsorbed onto the surfaces in the acidic pH region. As for the latter peptides, the amount adsorbed on the surface increased with increasing ionic strength. These results indicated that the carboxyl groups on the side chains of the peptides play an important role in the adsorption. Furthermore, the adsorption behavior of beta-lactoglobulin itself was found to be very similar to that of one of the latter peptides.

Structure-activity Relationship for the Insect Antifeedant Activity of Benzofuran Derivatives.

Coumaran (2,3-dihydrobenzofuran), a secondary metabolite of Cyperus nipponicus, inhibits the feeding of polyphagous insects. This secondary metabolite is regarded as one of the defensive systems of the Cyperaceae. A number of naturally occurring benzofurans that differ in their substitution pattern and oxidation state have been investigated for their ability to inhibit insect feeding by a bioassay with the common cutworm (Spodoptera litura F. Noctuidae) that applies the leaf disk method. The evaluation of the antifeedant activity of each test compound used the ED50 value based on the dose-response curve that was calculated with the probit method. The 2,3-dihydrobenzofuran derivative, 7-acetyl-4,6-dimethoxy-2-isopropenyl-2,3-dihydrobenzofuran, had an ED50 value of 1.3 µg (5.4×10(-9) mol)/cm(2) against the common cutworm. The introduction of methoxy and acetyl groups increased the insect antifeedant activity. Furthermore, the insect antifeedant activity increased with decreasing lipophilicity of the test compounds.

Regional differences in apoptosis in murine gliosarcoma (T9) induced by mild hyperthermia.

There are several reports of apoptosis induced by mild hyperthermia both in vitro and in vivo. However, in tumour nodules, regional differences in apoptosis induced by hyperthermia have not been studied morphologically or quantified. In this study, apoptosis was identified on the basis of its characteristic morphology, and was quantified in different tumour regions (margin and centre) by light and electron microscopy and discussed the correlation with necrosis. Apoptosis was induced in solid nodules of gliosarcoma (T9) in vivo by heating for 30 min in a water bath at 43 degrees C. The gliosarcoma cells showed a rapid increase in the number of apoptotic cells following mild hyperthermia. There was a regional difference in the rate of apoptosis immediately after hyperthermia. Necrosis was markedly enhanced only in the centre of the tumour 3 and 6 h after hyperthermia. These combined responses of apoptosis and necrosis seem to be caused by the microenvironmental change in the intratumour location.