[Use of sugammadex in a patient with limb girdle muscular dystrophy].

Limb girdle muscular dystrophy, a chronic progressive muscular atrophic disease, leads to high sensitivity to depolarizing and non-depolarizing neuromuscular blocking agents. We report the successful use of the sugammadex in a patient with limb girdle muscular dystrophy (dysferlinopathy, Miyoshi distal myopathy) to reverse rocuronium-induced neuromuscular block. After neuromuscular recovery to a train-of-four ratio = 43%, we administered 3.2 mg x kg(-1) of sugammadex (200 mg) intravenously, reversing neuromuscular blockade to a train-of-four ratio = 95% within 3 min. Sugammadex can be used to reverse rocuronium-induced neuromuscular blockade in patients with limb girdle muscular dystrophy.

[To distinguish true glottic opening using laryngoscope is very difficult for probationers].

BACKGROUND: The video intubating laryngoscope (VIL) can share information with co-workers, that otherwise only one probationer could obtain. Tracheal intubation was reviewed using recorded videotapes via VIL. It was supposed that the esophageal changes by laryngoscope might cause esophageal intubation. In this study, we investigated the impressions about the changed esophagus caused by laryngoscope, and our purpose is to find better educational method for tracheal intubation. METHODS: We randomly selected 21 first year junior residents for inexperienced group and 12 expert anesthesiologists for expert group, respectively. They were asked one questionnaire; "What do you think of it?" about three snapshots (A, B, C) recorded by VIL. Each answer was compared in both groups. RESULTS: The percentage of correct answers to snapshot A were 19% and 50% (P = 0.065) in inexperienced group and expert group; snapshot B, 14% and 50% (P = 0.026); and snapshot C, 14% and 92% (P < 0.0001), respectively. CONCLUSIONS: It was clarified that probationers significantly more often recognized the changed esophagus as the glottis opening. It is very important for us to teach and emphasize those points when we instruct the inexperienced in tracheal intubation.

[Study on the working conditions of rotating residents in the department of anesthesia under the new residency programs].

BACKGROUND: Jichi Medical University Hospital established a new residency program in 2004, which restricted the working time of junior residents to less than 80 hours for one week involving conferences and lectures. METHODS: We evaluated the working conditions of non-anesthesia residents in the department of anesthesia from April 2005 to March 2006. RESULTS: Non-anesthesia residents were engaged in anesthesia for 112 +/- 37 (mean +/-SD) hours for one month. CONCLUSIONS: Non-anesthesia residents worked in the anesthesia department under the regulation of the new residency program. However, a significant increase in the number of anesthesia cases, observed in recent years, is a potential imperilment of the new residency program.

Molecular, biochemical and immunological analyses of canine pancreatic DNase I.

The DNase I from canine pancreas was purified 260-fold to electrophoretic homogeneity with a 35% yield using three-step column chromatography. The activity of the purified enzyme was completely inhibited by 20 mM EDTA, an antibody specific to the purified enzyme and G-actin. A 1,373-bp cDNA encoding canine DNase I was constructed from the total canine pancreatic RNA using a rapid amplification of cDNA ends method, followed by sequencing. The mature canine DNase I protein was found to consist of 262 amino acids. A survey of DNase I in 13 different canine tissues revealed the highest levels of both DNase I enzyme activity and gene expression in the pancreas; therefore, the canine DNase I is of the pancreatic type. Phylogenetic and sequence identity analyses, studies of immunological properties and the tissue-distribution patterns of DNase I indicated that the canine enzyme is more closely related to the human DNase I than to other mammalian DNases I. Therefore, canine DNase I is found to be one of the best substitutes in studies of human DNase I.

Production of murine monoclonal anti-human DNase II antibodies, and their use for immunoaffinity purification of DNase II from human liver and urine.

Murine monoclonal anti-human deoxyribonuclease II (DNase II) antibodies were obtained from BALB/c mice immunized with human DNase II purified from human liver. Both single radial enzyme diffusion and DNA-cast polyacrylamide gel electrophoresis were very useful screening methods for obtaining the DNase II-specific antibodies. All of the antibodies showed specific inhibition of human DNase II enzyme activity and specific immunostaining. Insertion of the immunoaffinity step in our purification procedure made the purification of human DNase II easier, faster and more effective than the conventional procedure.

Carp hepatopancreatic DNase I: biochemical, molecular, and immunological properties.

A survey of DNase I in nine different carp tissues showed that the hepatopancreas has the highest levels of both DNase I enzyme activity and gene expression. Carp hepatopancreatic DNase I was purified 17,000-fold, with a yield of 29%, to electrophoretic homogeneity using three-step column chromatography. The purified enzyme activity was inhibited completely by 20 mM EDTA and a specific anti-carp DNase I antibody and slightly by G-actin. Histochemical analysis using this antibody revealed the strongest immunoreactivity in the cytoplasm of pancreatic tissue, but not in that of hepatic tissue in the carp hepatopancreas. A 995-bp cDNA encoding carp DNase I was constructed from total RNA from carp hepatopancreas. The mature carp DNase I protein comprises 260 amino acids, the same number as the human enzyme, however, the carp enzyme has an insertion of Ser59 and a deletion of Ala225 in comparison with the human enzyme. These alterations have no influence on the enzyme activity and stability. Three amino acid residues, Tyr65, Val67, and Ala114, of human DNase I are involved in actin binding, whereas those of carp DNase I are shifted to Tyr66, Val68, and Phe115, respectively, by the insertion of Ser59: the decrease in affinity to actin is due to one amino acid substitution, Ala114Phe. The results of our phylogenetic and immunological analyses indicate that carp DNase I is not closely related to the mammalian, avian or amphibian enzymes, and forms a relatively tight piscine cluster with the tilapia enzyme.

A single amino acid substitution of Leu130Ile in snake DNases I contributes to the acquisition of thermal stability.

We purified pancreatic deoxyribonucleases I (DNases I) from three snakes, Elaphe quadrivirgata, Elaphe climacophora and Agkistrodon blomhoffii, and cloned their cDNAs. Each mature snake DNase I protein comprised 262 amino acids. Wild-type snake DNases I with Leu130 were more thermally unstable than wild-type mammalian and avian DNases I with Ile130. After substitution of Leu130Ile, the thermal stabilities of the snake enzymes were higher than those of their wild-type counterparts and similar to mammalian wild-type enzyme levels. Conversely, substituting Ile130Leu of mammalian DNases I made them more thermally unstable than their wild-type counterparts. Therefore, a single amino acid substitution, Leu130Ile, might be involved in an evolutionally critical change in the thermal stabilities of vertebrate DNases I. Amphibian DNases I have a Ser205 insertion in a Ca2+-binding site of mammalian and avian enzymes that reduces their thermal stabilities [Takeshita, H., Yasuda, T., Iida, R., Nakajima, T., Mori, S., Mogi, K., Kaneko, Y. & Kishi, K. (2001) Biochem. J.357, 473-480]. Thus, it is plausible that the thermally stable wild-type DNases I of the higher vertebrates, such as mammals and birds, have been generated by a single Leu130Ile substitution of reptilian enzymes through molecular evolution following Ser205 deletion from amphibian enzymes. This mechanism may reflect one of the evolutionary changes from cold-blooded to warm-blooded vertebrates.

A simple method of DNA extraction and STR typing from urine samples using a commercially available DNA/RNA extraction kit.

We devised a simple DNA extraction procedure suitable for STR typing of urine sample. Use of a commercially available DNA/RNA extraction kit equipped with a silica-gel-based membrane made it possible to omit the recovery of urinary nucleated cells by sedimentation before the extraction. Successful genotyping of the TH01, HumTPO and multiplex STRs was achieved using aliquots of urine as small as 100 microL. Furthermore, application of this DNA extraction procedure to frozen urine samples provided STR allele results comparable to results obtained from fresh samples. Therefore, this extraction procedure is considered to be effective for STR typing of urine samples in both the frozen and aqueous state. Furthermore, addition of sodium azide to fresh urine samples prolonged their storage duration even at room temperature.

Production and characterization of murine monoclonal anti-human DNase II antibodies, and their use for immunoaffinity purification of DNase II from human liver and urine.

Four murine monoclonal anti-human deoxyribonuclease II (DNase II) antibodies were obtained from BALB/c mice immunized with human DNase II purified from human liver. Both single radial enzyme diffusion (SRED) and DNA-cast polyacrylamide gel electrophoresis (DNA-cast PAGE) were very useful for obtaining the DNase II-specific antibodies. All of the antibodies showed specific inhibition of human DNase II enzyme activity and specific immunostaining of the 32-kDa enzyme band, which is one of the three non-identical subunits of human DNase II molecule separated by sodium dodecyl sulfate (SDS)-PAGE followed by blotting on a transfer membrane. A formyl-cellulofine resin conjugated with each antibody specifically adsorbed and efficiently desorbed the active DNase II enzyme. Insertion of the immunoaffinity step in our purification procedure made the purification of human DNase II easier, faster and more effective than the conventional procedure.

Abrupt pubertal elevation of DNase I gene expression in human pituitary glands of both sexes.

Deoxyribonuclease I (DNase I) was confirmed to be expressed in the human pituitary gland, particularly the anterior lobe, at levels comparable to those in the pancreas. The DNase I activity and the amount of gene transcript present in the pituitary glands of individuals aged from 1 month to 89 years was significantly age-dependent, with an abrupt elevation after the neonatal and prepubertal periods irrespective of gender, followed by a gradual age-dependent decline in males and a marked reduction in females in their postreproductive period. This DNase I age dependence in the pituitary gland was not present in the pancreas and serum. These observations suggest that tissue-specific up-regulation of DNase I gene expression in the pituitary gland occur, possibly at the onset of puberty.