Marked lateral deviation of the phrenic nerve due to variant origin and course of the thyrocervical trunk: a cadaveric study.

Phrenic nerve impairment can often lead to serious respiratory disorders under various pathological conditions. During routine dissection of an 88-year-old Japanese male cadaver, a victim of heart failure, we recognized an extremely rare variation of the right thyrocervical trunk arising from the subclavian artery laterally to the anterior scalene muscle. In addition to that, the ipsilateral phrenic nerve was drawn and displaced remarkably laterad by this vessel. We examined all of the branches arising from subclavian arteries, phrenic nerves and diaphragm muscles. The embryological background of this arterial variation is considered. The marked displacement with prolonged strain had a potential to cause phrenic nerve impairment with an atrophic change of the diaphragm muscle. Recently many image diagnostic technologies have been developed and are often used. However, it is still possible that rare variations like this case may be overlooked and can only be recognized by intimate regional examination while keeping these rare variations in mind.

Japanese nurse practitioner practice and outcomes in a nursing home.

AIM: By describing the practice of a Japanese nurse practitioner, this descriptive case study discusses role development and outcomes before and after the intervention. BACKGROUND: One of the first Japanese nurse practitioners intervened at a nursing home during the government-designated trial period for nurse practitioner practice. CONCLUSION: Because of the nurse practitioner's meticulous observation and timely care provision to the residents in collaboration with the physician and the other staff in the facility, comparative data showed improvement in daily health status management of every resident and decreased deterioration of residents' health conditions requiring ambulance transfer and hospitalization.

Porphyromonas gingivalis C-terminal signal peptidase PG0026 and HagA interact with outer membrane protein PG27/LptO.

Outer membrane protein PG27 is essential for secretion/maturation of conserved C-terminal domain (CTD) proteins such as gingipains, HagA, and PG0026. To determine the binding partner(s) of PG27, we used a Porphyromonas gingivalis mutant strain, 83K48, which expressed functional histidine-tagged PG27. Purification of histidine-tagged PG27 from 83K48 found that 136-kDa and 264-kDa proteins accompanied histidine-tagged PG27. Mass spectrometry revealed the 136-kDa protein and 264-kDa protein to be PG0026 and PG1837 (HagA), respectively. PG0026 is a C-terminal signal peptidase which cleaves the CTDs of other CTD proteins. A cross-linking and a native electrophoresis studies suggested the interaction between histidine-tagged PG27 and HagA and the interaction between histidine-tagged PG27 and PG0026. We constructed Porphyromonas gingivalis gene disrupting mutants, and characterized them. PG0026 was required for the full activities of gingipains, whereas HagA was not. A mutation disrupting PG0026 affected localization of PG27, but a mutation disrupting PG1837 showed little effect on the expression and localizations of PG27 and PG0026. To determine the functional role of HagA, we used PG1837-disruptant 83K54 which expressed functional histidine-tagged PG27. Histidine-tagged PG27 in 83K54 was co-purified with not only PG0026 but also several contaminated proteins. These results suggest that PG0026 interacts with PG27 in the absence of HagA, and that the binding state of a PG27-PG0026 complex was affected and stabilized by HagA. Taken together, these data suggest that secreted PG0026 anchors to the cell by interacting with PG27, is stabilized by HagA, and functions in processing of other CTD proteins such as gingipains.

Analysis of Porphyromonas gingivalis PG27 by deletion and intragenic suppressor mutation analyses.

PG27 is required for secretion of virulence factor gingipains, and has recently been proposed as LptO, which is involved in O-deacylation of lipopolysaccharide. In the present study, a predicted 14 anti-parallel ß-strand structure of PG27 was ascertained. Deletion study showed that the region from Asp382 to the C-terminal His391 of PG27 is dispensable for the function of PG27. Analysis of C-terminal deletion mutants revealed that the region in strand S14 (Asn369-Gly385) is important for activity. Of the gingipain-defective mutants, ΔThr378-His391 and ΔPhe377-His391 produced amounts of PG27 comparable to those produced by wild-type cells, suggesting that Thr378-Phe381 contains essential residues for the function of PG27. In contrast, ΔPhe381-His391, ΔAla380-His391, ΔLeu379-His391 and ΔArg376-His391 produced no detectable PG27. The defects of the ΔAla380-His391 mutant were suppressed by changing either Ala346 or Ala359 of PG27 to valine. Importantly, Ala346 and Ala359 are located close to Leu379 in the structural model of PG27. A359V compensated for the instability of PG27, but not the gingipain-defective phenotypes, of other deletion mutants tested, suggesting that Ala380 and Phe381 of PG27 are important for the stability of PG27. Lastly, we found that the C-terminal region of PG27 may be located in the periplasm. Taken together, these findings fit well with a predicted ß-barrel structure model for PG27, and show that strand S14 is important for its function.

In vitro splicing analysis showed that availability of a cryptic splice site is not a determinant for alternative splicing patterns caused by +1G-->A mutations in introns of the dystrophin gene.

BACKGROUND: Splicing patterns are critical for assessing clinical phenotype of mutations in the dystrophin gene. However, it is still unclear how to predict alternative splicing pathways in such cases of splice-site mutation in the dystrophin gene. OBJECTIVE: To identify elements determining alternative splicing pathways in intron +1G-->A mutations of the dystrophin gene. RESULTS: We found that exon 25 is spliced out in the +1G-->A mutation in intron 25, resulting in mild Becker muscular dystrophy, and that a cryptic splice site within exon 45 was activated in severe Duchenne muscular dystrophy with a mutation of +1G-->A mutation in 45. Furthermore, in vitro splicing analysis using a pre-constructed expression vector showed that the mutant intron 25 produced one transcript that lacked exon 25. In contrast, the same splice-site mutation in intron 45 produced three splicing products. One product used the same cryptic donor splice site within exon 45 as the in vivo donor site and another product used a cryptic splice site within the vector sequence. Notably, the available cryptic splice site was not activated by the same G-->A mutation of intron 25. CONCLUSION: It was concluded that sequences inserted into the in vitro splicing assay minigene contain cis-elements that determine splicing pathways. By taking other +1G-->A mutations in the introns of the dystrophin gene reported in the literature into consideration, it seems that cryptic splice-site activation is seen only in strong exons. This finding will help to elucidate the molecular pathogenesis of dystrophinopathy and to predict efficiency of induction of exon skipping with antisense oligonucleotides for treatment of Duchenne muscular dystrophy.

Participation of the secreted dipeptidyl and tripeptidyl aminopeptidases in asaccharolytic growth of Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis secretes gingipains, endopeptidases essential for the asaccharolytic growth of this bacterium. P. gingivalis also secretes dipeptidyl aminopeptidases (DPPIV and DPP-7) and a tripeptidyl aminopeptidase (PTP-A), although their role in asaccharolytic growth is unclear. The present study was carried out to elucidate the role of these dipeptidyl/tripeptidyl aminopeptidases on the asaccharolytic growth of P. gingivalis. MATERIAL AND METHODS: Knockout mutants for the DPPIV (dpp), dpp7 and/or PTP-A genes were constructed. Brain-heart infusion medium supplemented with sterile hemin and menadione (BHIHM) was used as a complex medium, and the minimal medium used was GA, in which the sole energy source was a mixture of immunoglobulin G and bovine serum albumin. Growth of P. gingivalis was monitored by measuring the optical density of the culture. RESULTS: All knockout mutants for DPPIV, dpp7 and PTP-A grew as well as strain W83 in BHIHM. In GA, growth of single-knockout and double-knockout mutants was similar to that of W83, whereas growth of a triple-knockout mutant (83-47A) was reduced. We purified recombinant DPPIV and recombinant PTP-A from recombinant Escherichia coli overproducers, and purified DPP-7 from the triple-knockout mutant 83-4A. GA supplemented with the three purified dipeptidyl/tripeptidyl aminopeptidases supported the growth of 83-47A. CONCLUSION: DPPIV, DPP-7 and PTP-A contribute to the normal growth of P. gingivalis by cleaving substrate peptides into short-chain polypeptides that are efficient energy sources for P. gingivalis.

Effect of gamma-immunoglobulin on the asaccharolytic growth of Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVES: A minimal medium is indispensable for examining the growth properties of the asaccharolytic bacterium, Porphyromonas gingivalis. The purpose of the present study was to improve the widely used KGB medium to support good growth of P. gingivalis. MATERIAL AND METHODS: Growth of P. gingivalis (W50, W83, and ATCC33277) in a minimal medium was monitored by measuring the optical density of the culture during incubation. RESULTS: W50, W83, and ATCC33277 grew poorly with bovine serum albumin as the sole carbon and nitrogen source, and alpha-ketoglutarate had little or no effect on this poor growth. In contrast, FeCl3 improved the growth of W83 and ATCC33277; however, the use of a high concentration of FeCl3 elicited black pigmentation of the cells. Bovine gamma-immunoglobulin greatly recovered the growth defect. None of alpha-ketoglutarate, citrate, or trace metal ions, when used to supplement KGB medium, was required for growth. We determined the optimal conditions for growth, and developed a new simple minimal medium for P. gingivalis (GA medium). Growth of ATCC33277 in GA medium was dependent on gingipains; Arg-gingipains and Lys-gingipain contributed comparably to proliferation of the bacterium. CONCLUSION: These data indicate that GA medium is currently the most reliable minimal medium for examining the growth properties of P. gingivalis.

Time-dependent changes in the carotenoid composition and preferential binding of spirilloxanthin to the reaction center and anhydrorhodovibrin to the LH1 antenna complex in Rhodobium marinum.

Carotenoids were isolated from the cells of Rhodobium marinum, and their structures were determined by mass spectrometry and 1H nuclear magnetic resonance spectroscopy; the carotenoids include lycopene, rhodopin, anhydrorhodovibrin, rhodovibrin and spirilloxanthin. Time-dependent changes in the carotenoid composition in the reaction center (RC) and the light-harvesting complex 1 (LH1) were traced by high-performance liquid chromatography analysis of the extracts. The carotenoid composition changed according to the spirilloxanthin biosynthetic pathway. However, spirilloxanthin having the longest conjugated chain was always preferentially bound to the RC, and anhydrorhodovibrin and other precursors to the LH1.

Development of an enzyme immunoassay for serum 16-dehydropregnenolone.

We have developed an enzyme immunoassay (EIA) for serum 16-dehydropregnenolone (3beta-hydroxy-5,16-pregnadien-20-one; 16-DHP). The antiserum against 16-DHP-3-hemisuccinate conjugated bovine serum albumin (16-DHP-3HS-BSA) was raised in rabbits. For use as an enzyme labeled antigen, 16-DHP-3HS was conjugated to alkaline phosphatase. The minimal amount of 16-DHP detected was 4 pg (0.013 pmol)/assay and the measurable range was from 0.06-60 ng/ml (0.191-191 nmol/l). The intra-assay coefficient of variation (C.V.) was 4.1% (0.73+/-0.03 ng/ml, mean+/-S.D., n=6), and inter-assay C.V. was 7.7% (0.13+/-0.01 ng/ml, n=6). A liner relation was observed between the serum sample dilution and the 16-DHP concentration. For the recovery study, authentic 16-DHP was added to a serum sample (original concentration: 0.10-0.14 ng/ml), and the recovery was found to be 94.4-96.8% (final 16-DHP concentrations calculated: 0.29-16.3 ng/ml). To investigate the reliability of the present EIA, the values from our EIA were compared with those obtained by GC-MS. The 16-DHP concentration could not be measured in serum by GC-MS because of its sensitivity. Therefore, the conjugated steroid, 16-DHPS, was first enzymatically hydrolysed and then the 16-DHP measured by both methods. There was a good correlation between the levels determined by these methods (Pearson's correlation coefficient: r=0.927, p<0.001, y=0.74x+3.61, n=27). The serum concentrations of 16-DHP in neonates and umbilical vein were 0.53+/-0.09 ng/ml and 0.88+/-0.61 ng/ml, respectively. No 16-DHP was detected in serum from normal healthy adults using the present EIA. These results suggest that 16-DHP originates from the fetus and neonate.

Reconstitution and purification of cytolethal distending toxin of Actinobacillus actinomycetemcomitans.

Cytolethal distending toxin (CDT) has been found in various pathogenic bacterial species and causes a cell distending and a G2 arrest against eukaryotic cells. All the cdtABC genes, which encode CDT, are known to be required for the CDT activities although the CDT holotoxin structure has not been elucidated. We cloned the cdtABC genes of Actinobacillus actinomycetemcomitans and constructed an Escherichia coli expression system for them. We found that crude extracts from six deletion mutants (delta cdtA, delta cdtB, delta cdtC, delta cdtBC, delta cdtAC, and delta cdtAB) of recombinant E. coli, which showed very weak or no detectable CDT activities, restored the CDT activities when pre-mixing and pre-incubation of them were performed in combinations to contain all the CdtA, CdtB, and CdtC proteins. These results indicate that all the Cdt proteins are required for the CDT activities. We also found that the chimera CdtB protein, CdtB-intein-CBD (chitin binding domain) like CdtB protein itself assembled with CdtA and CdtC. The reconstituted CDT containing the chimera CdtB protein was specifically extracted by chitin beads and the only CDT portion was isolated from the chitin beads by a cleavage reaction of the intein. The purified reconstituted-CDT was found to consist of CdtA, CdtB, and CdtC proteins, and showed appreciable CDT activities, indicating that the CDT holotoxin structure is the CdtABC complex. To our knowledge, this is the first report succeeded in complete purification of an active CDT and may offer useful tools for elucidation of the toxic mechanism of CDT.

Staged spinal cord decompression through posterior approach for thoracic myelopathy caused by ossification of posterior longitudinal ligament.

STUDY DESIGN: Prospective clinical study of the effect of staged elimination of anatomic factors inhibiting posterior shift of the thoracic spinal cord on the degree of posterior shift of the thoracic spinal cord and its significance in augmenting the safety of ossification of posterior longitudinal ligament (OPLL) manipulation in thoracic OPLL myelopathy. OBJECTIVES: To develop a comprehensive method that enables safe and sufficient decompression of the spinal cord for thoracic OPLL myelopathy. SUMMARY OF BACKGROUND DATA: Decompression of the spinal cord by direct manipulations of thoracic OPLLs, via either anterior or posterior approach, caused some iatrogenic catastrophic spinal cord injuries, and methods to prevent such injuries during surgery have not yet been developed. METHODS: Procedures of elimination of anatomic factors inhibiting posterior shift of the thoracic spinal cord were performed in stages at intervals of between 1 month and 11 years depending on patients' neurologic status. The first stage operation consisted of extensive cervicothoracic laminoplastic decompression with or without posterior longitudinal durotomy, and if the decompression were insufficient, measures for OPLL-spinal cord separation with or without OPLL manipulation were added. RESULTS: All 17 patients with thoracic OPLL myelopathy showed improvements of neurology comparable with those with successful anterior approaches after decompression. The mean follow-up period was 42 months (range 6-101 months). Neurologic improvements persisted for the entire follow-up period in all patients except one patient who developed arachnoid cyst compressing the dorsum of the once-decompressed spinal cord 30 months after surgery. CONCLUSIONS: Staged posterior decompression to eliminate anatomic factors inhibiting posterior shift of the thoracic spinal cord is the safest and the most reliable method of spinal cord decompression to treat thoracic OPLL myelopathy, so far. However, long-term results are required before the methods can be established.

Specific determination of urinary 6beta-hydroxycortisol and cortisol by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

The urinary 6beta-hydroxycortisol to cortisol ratio is believed to be a noninvasive index of cytochrome P450 3A activity. For precise assessment of the ratio in human urine, we have developed a reversed-phase high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry method. The selective method was accurate and reproducible with intra- and inter-day precision of variation coefficients of less than 8%. The 6beta-hydroxycortisol to cortisol ratio ranged from 3.0 to 12.4 in healthy Japanese 24-h urine. With the recent popularization of LC-MS, our LC-MS method will be advantageous to detect human in vivo CYP3A activity for clinical investigation and routine measurement in various laboratories.

The value of a new method for assessing the separate functions of the long tracts and involved segments in patients with cervical myelopathy.

In order to assess accurately lesions of the spinal cord in patients with cervical myelopathy we have developed a new method of examination, which is based on the Japanese Orthopaedic Association (JOA) scoring system. The method attempts to assess separately the functions of the long tract and any involved cord segments in respect to the period after treatment. It was used in 117 consecutive patients who were divided into 2 groups based on whether or not there was a T2-high-intensity lesion within the spinal cord, as revealed by a preoperative magnetic resonance imaging scan (MRI). The results of this method correlated well with the MRI findings. It was assumed that the degree of function of the upper limbs in patients with a T2-high-intensity lesion revealed more about a segment than about the long tract.

Diffusion-weighted echo planar imaging of the normal human cervical spinal cord.

We obtained diffusion-weighted echo planar images of the human cervical cord in vivo and correlated them with histopathologic findings. Images were obtained in 17 healthy volunteers using a 1.5 T clinical MR unit. When motion-probing gradients were added perpendicular to the long axis of the cord, the white matter was hyperintense because of anisotropic diffusion. However, the gracile fasciculus was hypointense probably due to the small diameter of neural fibers and the large extracellular space.

[Cranial nonmetric variation of Yayoi people in the Kyushu District].

The Yayoi people in Kyushu and Yamaguchi area are generally classified by metrical analyses mainly into the Yayoi people in the northern Kyushu and Yamaguchi area who are regarded as migrants from the Asian Continent and their posterity and the Yayoi people in the northwestern Kyushu who are regarded as having inherited the characteristics of the Jomon people. Such classification is verified by the analysis with the 22 traits in the cranial nonmetric variation. Of the 22 traits, supraorbital foramen, transverse zygomatic suture vestige, biasterionic suture vestige, jugular foramen bridging, hypoglossal foramen bridging, pterygospinous foramen, mylohyoid bridging and tympanic dehiscence are particularly important as the traits to classify two types of Yayoi peoples. The analysis by the C. A. B. Smith's Mean Measure of Divergence (MMD) suggests that out of the migrant Yayoi peoples, the very Yayoi people who are closely related to the formation of the modern Japanese are the ones in the northern Kyushu area, not the ones in the Doigahama site. Also, it is assumed that the appearance and disappearance of cranial nonmetric variations is affected by genetic elements, because the incidencies of cranial nonmetric variations is largely different between two types of Yayoi peoples in infants like in adults. Lastly the cranial nonmetric variation in the people of the period equivalent to Jomon and Yayoi in the Okinawa district, and in the Kofun people in southern Kyushu area, was briefly introduced.

Circadian variation of the urinary 6beta-hydroxycortisol to cortisol ratio that would reflect hepatic CYP3A activity.

OBJECTIVES: Chronopharmacokinetics of drugs metabolized by cytochrome P450 3A (CYP3A) has been reported recently; however, little is studied on intra-individual circadian variation in CYP3A activity in human. The aim of this study was to assess the intra-individual diurnal variation and day-to-day variation of the urinary 6beta-hydroxycortisol to cortisol ratio, a noninvasive index of human CYP3A activity. METHODS: Urine samples from ten healthy Japanese men were collected over four time intervals (0900 hours to 1300 hours, 1300 hours to 1700 hours, 1700 hours to 2100 hours and 2100 hours to 0900 hours) on days 1, 5 and 14 to verify diurnal variation, and 24-h urine was collected to study day-to-day variation over 2 weeks. Urinary 6beta-hydroxycortisol and cortisol were determined by means of high-performance liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry. RESULTS: The ratio of urinary 6beta-hydroxycortisol to cortisol exhibited noteworthy diurnal variation intra-individually; 2.8-fold on average. However, day-do-day intra-individual variation of the ratio was not observed over 2 weeks; the coefficient of variation was 11.9 +/- 3.0%. CONCLUSION: The result indicates that imprudent use of random urine has a great risk of false evaluation in assessment of the 6beta-hydroxycortisol to cortisol ratio and that the ratio in 24-h urine samples provides a more robust measure of the inter-individual difference of this metabolic ratio, which to a certain but not complete extent represents the CYP3A activity.

Molecular identification and characterization of novel membrane-bound metalloprotease, the soluble secreted form of which hydrolyzes a variety of vasoactive peptides.

One class of zinc metalloproteases, represented by neutral endopeptidase 24.11 and endothelin-converting enzyme, has been shown to be involved in proteolytic activation or inactivation of many regulatory peptides. Here, we report molecular cloning and characterization of a novel member of this type II membrane-bound metalloprotease family, termed soluble secreted endopeptidase (SEP). Alternative splicing results in the generation of another transcript, SEP(Delta), which lacks a 69-base pair nucleotide segment following the transmembrane helix. Both SEP and SEP(Delta) mRNA are detected in all mouse tissues examined. Transfection of an SEP cDNA expression construct resulted in the expression of the membrane-bound form of SEP in the early secretory pathway as well as the soluble secreted form of the enzyme in the culture medium. In contrast, transfection of the SEP(Delta) cDNA only results in the expression of the membrane-bound form. In vitro enzymological analysis of the recombinant soluble form of SEP demonstrated that it hydrolyzes a variety of vasoactive peptides, including endothelin-1, atrial natriuretic peptide, and angiotensin I. This activity of SEP was inhibited by phosphoramidon and the neutral endopeptidase 24.11 specific inhibitor thiorphan, but it was only partially inhibited by the endothelin-converting enzyme specific inhibitor FR901533. These findings suggest that SEP is a novel metalloprotease that possesses a broad substrate specificity and that it may be involved in the metabolism of biologically active peptides intracellulary as well as extracellularly.

Determination of 1-methyl-1,2,3,4-tetrahydroisoquinoline in mouse brain after treatment with haloperidol by gas chromatography-selected ion monitoring.

The content of the endogenous amine, 1-methyl-1,2,3,4-tetrahydroisoquinoline (1-MeTIQ), in mouse brain, treated with the antipsychotic agent haloperidol (HP) was determined by GC-SIM (selected ion monitoring) system. 1-MeTIQ in brain was extracted with chloroform at pH 11-12 and was detected as PFP derivative by GC-SIM. The 1-MeTIQ contents in mouse brains following intraperitoneal administration of HP or its dehydrated product, HPTP (1 and 4 mg/kg per day, for four days), were markedly reduced compared with control groups. This result agrees well with the findings in human idiopathic parkinsonianism and in MPTP-treated mouse brain. In addition, this finding suggests that the change of the endogenous amine 1-MeTIQ content in the brain plays an important role in the pathogenesis of toxin-induced parkinsonism.

The effect of muscle-tone-reducing procedures in athetotic head movements: partial nerve block by lidocaine and surgical release of the neck muscles.

OBJECTIVE: To investigate the effect of muscle-tone-reducing procedures (MTRPs), i.e. partial nerve block by lidocaine (PNB) and surgical release of muscle attachment to bone (SRMAB), on incessant involuntary head movements in athetotic patients. METHODS: Pre/post-MTRP changes in neck-muscle activities, glabella movement and maximum isometric forces of the head were measured in six athetotic patients with severe spondylotic myelopathy resulting from incessant involuntary head movements. RESULTS: Pre/post-MTRP changes in neck-muscle activities resembled those after gamma-block. In four patients, PNB reduced the maximum isometric force by no more than 40% of pre-PNB force, while decreasing the amount of involuntary head movements to 37-65% of the pre-PNB value in the frontal plane. MRSAB reduced the force by less than 40% of pre-SRMAB force in 4 MRSAB tested patients, while decreasing the amount of involuntary head movements to 12-45% of the pre-SRMAB value in all 6 patients. CONCLUSION: MTRPs reduced involuntary head movements significantly while preserving voluntary muscle forces relatively well. PNB and SRMAB procedures have in common the effect of reducing gain in the myotatic reflex pathway by decreasing the excitatory inflows to alpha-motoneurons via muscle spindle Ia-afferents, which resulted from blocking mainly gamma-efferent conduction by PNB, and reducing background tension in muscle spindles by SRMAB.