A planning target volume margin formula for hypofractionated intracranial stereotactic radiotherapy under cone beam CT image guidance with a six-degrees-of-freedom robotic couch and a mouthpiece-assisted mask system: a preliminary study.

OBJECTIVE: A planning target volume (PTV) margin formula for hypofractionated intracranial stereotactic radiotherapy (SRT) has been proposed under cone beam CT (CBCT) image guidance with a six-degrees-of-freedom (6-DOF) robotic couch. METHODS: CBCT-based registration using a 6-DOF couch reportedly led to negligibly small systematic positioning errors, suggesting that each in-treatment positioning error during the treatment courses for the patients employing this combination was predominantly caused by a random gaussian process. Under this assumption, an anisotropic PTV margin for each axis was formulated based on a gaussian distribution model. 19 patients with intracranial lesions who underwent additional post-treatment CBCT were consecutively selected, to whom stereotactic hypofractionated radiotherapy was delivered by a linear accelerator equipped with a CBCT imager, a 6-DOF couch and a mouthpiece-assisted mask system. Time-averaged patient-positioning errors during treatment were estimated by comparing the post-treatment CBCT with the reference planning CT images. RESULTS: It was suggested that each histogram of the in-treatment positioning error in each axis would approach each single gaussian distribution with a mean of zero. The calculated PTV margins in the x, y and z directions were 0.97, 1.30 and 0.88 mm, respectively. CONCLUSION: The empirical isotropic PTV margin of 2 mm used in our facility for intracranial SRT was consistent with the margin calculated by the proposed gaussian model. ADVANCES IN KNOWLEDGE: We have proposed a PTV margin formula for hypofractionated intracranial SRT under CBCT image guidance with a 6-DOF robotic couch.

Enhancement of immunoregulatory effects of Lactobacillus gasseri TMC0356 by heat treatment and culture medium.

AIMS: The aim of this study was to investigate the influence of heat treatment and culture media on the immunoregulatory effects of a probiotic strain, Lactobacillus gasseri TMC0356 (TMC0356). METHODS AND RESULTS: TMC0356 cultured in deMan-Rogosa-Sharpe and same food grade (FG) media were inactivated with the heat treatment at 70 and 90°C. Viable and heat-killed TMC0356 were tested for their ability to induce interleukin (IL)-12 production in the murine macrophage cell line J774.1. These TMC0356 were examined for their resistance to N-acetylmuramidase. Their morphology was observed by scanning electron microscopy. The heat-killed TMC0356 significantly induced IL-12 production in J774.1 cells and exhibited enhanced resistance to N-acetylmuramidase compared with viable TMC0356. Morphological changes were observed in TMC0356 when cultured in FG medium. Cell morphology and induction of IL-12 production in J774.1 cells were also associated. CONCLUSIONS: These results suggest that heat treatment and culture medium composition modified the immunoregulatory effects of TMC0356 to induce IL-12 production in macrophages. SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrate that probiotic immunoregulatory effects may be modified by the processing technology of cell preparation.

Dose verification of intensity-modulated arc therapy using an ERGO++ treatment planning system and Elekta internal multileaf collimators for prostate cancer treatment.

Dose verification of intensity-modulated arc therapy using an ERGO++ treatment planning system and Elekta internal multileaf collimators is described. Prostate intensity-modulated arc therapy was planned using the arc modulation optimization algorithm inverse planning module of ERGO++. After transferring the plan to Elekta Synergy's controller (Elekta Ltd, Crawley, UK), the isocentre dose was measured and compared with a calculated dose using a pinpoint chamber and a water phantom in a cylindrical acrylic enclosure. Subsequently, an EDR2 film was placed inside a multilayer plastic phantom, and total dose distributions were measured in three axial planes as well as in the coronal and sagittal planes to compare the actual dose with the calculated dose. The dose discrepancy at the isocentre was 1.7%. The calculated gamma indices were less than 1 over 90% of the three axial planes, as well as in the coronal and sagittal planes, having a dose greater than 50% of the maximum target dose.

Mutations that are synthetically lethal with a gas1Delta allele cause defects in the cell wall of Saccharomyces cerevisiae.

The GAS1-related genes of fungi encode GPI-anchored proteins with beta-1,3-glucanosyltransferase activity. Loss of this activity results in defects in the assembly of the cell wall. We isolated mutants that show a synthetic defect when combined with a gas1Delta allele in Saccharomyces cerevisiae, and identified nine wild-type genes that rescue this defect. The indispensability of BIG1 and KRE6 for the viability of gas1Delta cells confirmed the important role of beta-1,6-glucan in cells that are defective in the processing of beta-1,3-glucan. The identification of the Wsc1p hypo-osmotic stress sensor and components of the PKC signal transduction pathway in our screen also confirmed that the cell wall integrity response attenuates the otherwise lethal gas1Delta defect. Unexpectedly, we found that the KEX2 gene is also required for the viability of the gas1Delta mutant. Kex2p is a Golgi/endosome-membrane-anchored protease that processes secretory preproteins. A cell wall defect was also found in the kex2Delta mutant, which was suppressible by multiple copies of the MKC7 or YAP3 gene, both of which encode other GPI-anchored proteases. Therefore, normal cell wall assembly requires proteolytic processing of secretory preproteins. Furthermore, the genes CSG2 and IPT1 were found to be required for normal growth of gas1Delta cells in the presence of 1 M sorbitol. This finding suggests that complex sphingolipids play a role in the hyper-osmotic response.

A method for achieving variable widths of the spread-out Bragg peak using a ridge filter.

A method for designing a variable-SOBP (spread-out Bragg peak) ridge filter has been proposed. First, ridge filter parameters are determined by using a Monte Carlo calculation followed by a fast two-step iterative optimization. Then, tilting the ridge filter results in continuous variation of the SOBP width. Monte Carlo calculations show that depth dose uniformity changes from +/- 1.3% to +/- 1.6% for SOBP widths ranging from 10.3 cm to 14.5 cm. Advantages of the proposed tilting ridge filter include a capability of continuous SOBP variation and cost-effective installation for a given SOBP width range.

Molecular characterization of a novel yeast cell-wall acid phosphatase cloned from Kluyveromyces marxianus.

A novel Kluyveromyces marxianus gene that encodes an acid phosphatase, Pho610, was cloned in Saccharomyces cerevisiae. The deduced amino acid sequence was distinct from S. cerevisiae phosphatases but similar to some fungal enzymes. A peculiar feature of the sequence is that it has hydrophobic stretches both at the N- and C-termini, which is a characteristic of the precursors of glycosylphosphatidylinositol(GPI)-anchored proteins. When the gene was expressed in S. cerevisiae, the active enzyme was recovered in the periplasmic fraction by glucanase digestion. The Pho610 polypeptide was highly glycosylated and a significant portion was covalently linked to the cell-wall glucan. The enzyme was secreted when the C-terminal region was truncated to remove the GPI signal. Therefore, Pho610 is a novel cell-wall protein having an enzyme activity.

Proteins in the early golgi compartment of Saccharomyces cerevisiae immunoisolated by Sed5p.

The yeast tSNARE Sed5p is considered to mainly reside in the early Golgi compartment at the steady state of its intracellular cycling. To better understand this compartment, we immunoisolated a membrane subfraction having Sed5p on the surface (the Sed5 vesicles). Immunoblot studies showed that considerable portions (20-30%) of the Golgi mannosyltransferases (Mnt1p, Van1p, and Mnn9p) were simultaneously recovered while the late Golgi (Kex2p) or endoplasmic reticulum (Sec71p) proteins were almost excluded. The N-terminal sequences of the polypeptides detectable by Coomassie blue staining indicated that the prominent components of the Sed5 vesicles include Anp1p, Emp24p, Erv25p, Erp1p, Ypt52p, and a putative membrane protein of unknown function (Yml067c).

Oropharyngotonsillitis associated with nonprimary Epstein-Barr virus infection.

OBJECTIVE: To identify distinct clinical features of pharyngotonsillitis or oropharyngitis associated with Epstein-Barr virus (EBV) infection from herpes simplex virus infection. DESIGN: Clinical studies by case exploration. SETTING: Institutional practice at a university hospital. PATIENTS: Thirty-three patients with pharyngotonsillitis and 4 patients with oropharyngitis of nonbacterial infection underwent biopsy of pharyngotonsillar lesions. MAIN OUTCOME MEASURE: The specimens were examined by histopathology, immunohistochemistry, in situ hybridization, and polymerase chain reaction. In addition to serological testing and routine laboratory data, photographic oropharyngeal findings were collected for clinical evaluation. RESULTS: In situ hybridization to detect EBV-encoded small nuclear RNA-1 and -2 disclosed 8 cases of pharyngotonsillitis and 4 cases of oropharyngitis associated with EBV infection. Immunohistochemical analysis identified 5 cases of pharyngotonsillitis associated with herpes simplex virus infection. Serological examination showed that, among 12 cases positive by in situ hybridization, 3 cases were primary infection with infectious mononucleosis and 9 were nonprimary infection. The staining pattern of in situ hybridization was different, ie, a linear pattern in cases of nonprimary infection and a scattered pattern in cases of primary infection. The clinical manifestations of EBV pharyngotonsillitis were distinct from those of herpes simplex virus pharyngotonsillitis and were characteristic irrespective of infectious status, while those of EBV oropharyngitis were more variable. CONCLUSIONS: Epstein-Barr virus-associated pharyngotonsillitis was demonstrated in patients with nonprimary infection unaccompanied by infectious mononucleosis. Epstein-Barr virus should be considered a potential causative agent of oropharyngotonsillitis even in absence of infectious mononucleosis, especially in a young adult.

High prevalence of human papillomaviruses in the normal oral cavity of adults.

Human papillomavirus (HPV) infection in the normal oral cavity was studied by the sensitive polymerase chain reaction (PCR) using primers for the L1 region of human papillomavirus DNA and high fidelity amplification system. Cells were scraped from the oral mucosae of 7 (mean age; 42 years) and 30 (mean age; 32 years) volunteers with and without skin warts, respectively. Human papillomavirus DNA was detected in 30/37 (81.1%) specimens and their copy numbers per cell were 10(-1) to 10(-4) (mean, 10(-3)). The human papillomavirus types determined by PCR-based sequencing analysis were HPV-18 (26/30; 86.7%), -61 (18/30; 60%), -59 (7/30; 23.3%), -16 (2/30; 6.7%), -6 (1/30; 3.3%) and an unknown type (HPV-X71) (1/30; 3.3%). Multiple human papillomavirus types were present in 17/30 (56.7%) specimens. HPV-6 was detected in 2 of 7 skin warts and differed from the human papillomavirus types of the corresponding oral specimens. These data suggest that human papillomavirus infection in the oral mucosa occurs much more frequently than previously considered.

Separation of a process from the neuronal cell body using a thin aluminum foil separator.

A new technique using a simple-structured separator is described to separate the end of the process from the cell body (soma) of cultured cells. By this method, stimulation limited to neuronal processes is experimentally possible. The separator is of an oblong shape and consists of two parts: a square bracket-shaped frame made of Teflon and a thin aluminum foil which is stuck on both free ends of frame (bracket) coated with epoxy-resin. The separator is horizontally placed on the dish under microscope to divide the process on the way, so that the free end of the process is outside the oblong separator. This separator is simple and low cost, and does not need a long process, unlike other methods requiring a special culture dish with a micro-groove for growth of the neuronal process (neurite). The new technique could be useful in basic pain research and neurophysiology.

Isolation and characterization of nickel-accumulating yeasts.

We selected three yeast strains that efficiently remove heavy metal ions from aqueous solution. We first screened yeasts that grew in the presence of 2 mM NiCl2 among our stock of wild yeasts, and then selected those that removed Ni most efficiently from aqueous solution. These strains also removed Cu and Zn from aqueous solution and were identified as Candida species. Ni uptake was efficient at pH between 4.0 and 7.0, but less efficient at pH below 3.0. The amount of Ni taken up by the yeast cells was proportional to the initial concentration of NiCl2 below about 4 mM Ni. The cells retained the abilities to remove Ni after treatment with 10 mM EDTA or 1 M HCl for repeated usage, or after heat treatment.

[The effect of low-dose prostaglandin E1 on serum and urinary fluoride concentrations in patients anesthetized with sevoflurane].

We investigated the effects of low-dose prostaglandin E1 (PGE1) on serum and urinary concentrations of inorganic fluoride in 39 adult patients undergoing upper abdominal surgery. Anesthesia was maintained with a combination of N2O-O2-sevoflurane and thoracic epidural anesthesia. Twenty-two patients received infusion of PGE1 at a rate of 0.02 micrograms.kg-1.min-1 throughout surgery. Seventeen patients served as control by not receiving PGE1. Serum inorganic fluoride concentrations (FB) were determined before the induction of anesthesia and 0, 2 and 24 hours after the end of anesthesia. Urinary inorganic fluoride concentrations (FU) were determined before the induction of anesthesia, and 0, 24 and 48 hours after the end of anesthesia. These was no difference between PGE1 group and control group in anesthetic dose (MAC hours) of sevoflurane. In both groups, FB peaked at the end of anesthesia. In PGE1 group, UB peaked at the end of anesthesia, while in control group, it peaked 24 hours after anesthesia. There were differences between groups neither in FB nor in FU throughout the study period. The relationships between anesthetic dose and fluoride concentrations, however, differed significantly between the groups. In control group FB values of 0, 2 and 24 hours after anesthesia correlated positively with MAC hours, respectively, while in PGE1 group they did not. Similarly in control group, FU values of 24 and 48 hours after anesthesia correlated positively with MAC hours, respectively, while in PGE1 group, they did not. Thus in patients receiving high-dose sevoflurane, FB and FU tended to be lower in PGE1 group than in control group. In contrast, in PGE1 group, urinary excretion of fluoride during surgery correlated positively with MAC hours, while in control group, it did not. Urinary fluoride excretion during surgery was significantly greater in PGE1 group than in control group. These results suggested that PGE1 might prevent elevation of serum and urinary fluoride concentrations in patients receiving high-dose sevoflurane. This effect might result from enhanced urinary excretion of fluoride with PGE1.

Novel membrane protein complexes for protein glycosylation in the yeast Golgi apparatus.

Three type II membrane proteins Anp1, Van1 and Mnn9 of Saccharomyces cerevisiae share significant sequence homology. Their precise biochemical activity has long been unknown though the mutant phenotype indicates their participation in protein glycosylation in the Golgi apparatus. To shed light on their molecular characteristics, interactions of these proteins were studied by immunoprecipitation after solubilizing the membrane by nonionic detergent. Our results indicated that there are at least two submembrane complexes containing these proteins: one contains Van1 and Mnn9 proteins and the other contains Anp1 and Mnn9 proteins. In addition, Hoc1 protein which has significant homology to Och1 protein colocalized with Anp1 and Mnn9 proteins. These complexes with similar but partially different constituents may represent essential parts of glycosylation machinery in the yeast Golgi compartments.

Dose optimization of proton and heavy ion therapy using generalized sampled pattern matching.

We have proposed a new dose optimization method for proton and heavy ion therapy using generalized sampled pattern matching, where an optimal beam weight distribution for scanning is obtained as a solution. Using water phantom models, one-dimensional lateral and depth dose distributions were separately optimized, each resulting in a uniform dose distribution within a target region and minimum dose fall-off to minimize undesired irradiation onto neighbouring tissues. Subsequently, we have applied the technique to broad beam three-dimensional proton therapy, leading to a homogeneous dose distribution inside a target and minimized distal and lateral dose fall-off for most convex tumour shapes.

Alteration of cell cycle-dependent histone phosphorylations by okadaic acid. Induction of mitosis-specific H3 phosphorylation and chromatin condensation in mammalian interphase cells.

Effects of okadaic acid (OA), a protein phosphatase inhibitor, on chromatin structure and phosphorylation of histones were examined using HeLa and N18 cells. The chromatin condensation in HeLa cells was mild and resemble prometaphase nuclei, while the condensation in N18 cells was extensive and chromatin became a compact body. H2A in HeLa cells was extensively and consistently phosphorylated at the same site throughout the cell cycle, and H3 was demonstrated to be phosphorylated at the mitosis-specific site Ser10. In contrast, H1 phosphorylation was rapidly decreased in most sites within 3 h. The reduction of H1 phosphorylation was accompanied by a quantitative change in the set of H1 phosphopeptides. During the early phase of the OA treatment, H1 phosphorylation was transiently elevated in tandem, whereas H3 phosphorylation reached a maximum somewhat later. The results suggest that mitosis-specific events (cdc2/H1 kinase activation, H1 superphosphorylation, mitosis-specific H3 phosphorylation and chromatin condensation) induced by OA are sequentially associated. The changes appear to reflect a molecular mechanism similar to that operating in normal mitosis.

Secretion of active subtilisin YaB by a simultaneous expression of separate pre-pro and pre-mature polypeptides in Bacillus subtilis.

Alkaline elastase YaB, produced by alkalophilic Bacillus YaB, is an extracellular serine protease having 55% homology to subtilisin BPN' and thus could be called subtilisin YaB. It is synthesized as a 378-amino acid preproenzyme and secreted into the culture medium as a 265-amino acid mature protease. To examine if the pro-peptide of subtilisin YaB functions in trans to guide the folding of secreted subtilisin YaB in vivo, we made genes encoding the pre-pro, pro and pre-mature portions and placed them under the control of the spac-1 promoter on a multi-copy plasmid. When simultaneous expression in Bacillus subtilis of both the pre-pro and pre-mature genes was induced with 0.5 mM isopropyl-1-thio-beta-D-galactopyranoside (IPTG), protease activity was detected in the medium. On the other hand, we could not detect protease activity when the expression of either the pre-mature gene alone or both the pro and pre-mature genes was induced. From these results, we concluded that the pro region functions in trans and outside the cells for the proper folding and activation of the enzyme.

[Management of epidural anesthesia in a patient with aortitis syndrome].

We report an anesthetic management of a patient with severe vascular occlusion case caused by Aortitis syndrome. The patient, 61 yr-old woman, was scheduled for sigmoidectomy. Her bilateral common carotid arteries and left main coronary artery were totally occluded, but she had been asymptomatic for over thirty years. Epidural anesthesia was chosen to provide pain control, cardiovascular stability, and also to maintain consciousness during operation for cerebral circulatory evaluation. Following intravenous administration of lactated Ringer's solution (about 1000 ml), epidural blockade was maintained with 1 to approximately 1.5% lidocaine and butorphanol. Our findings in this case indicate that it is important for coronary and cerebral perfusion to have normal central venous pressure (preload) and arterial pressure during sympathetic blockade resulting from epidural anesthesia.

bfr1+, a novel gene of Schizosaccharomyces pombe which confers brefeldin A resistance, is structurally related to the ATP-binding cassette superfamily.

We have isolated a Schizosaccharomyces pombe gene, bfr1+, which on a multicopy plasmid vector, pDB248', confers resistance to brefeldin A (BFA), an inhibitor of intracellular protein transport. This gene encodes a novel protein of 1,531 amino acids with an intramolecular duplicated structure, each half containing a single ATP-binding consensus sequence and a set of six transmembrane sequences. This structural characteristic of bfr1+ protein resembles that of mammalian P-glycoprotein, which, by exporting a variety of anticancer drugs, has been shown to be responsible for multidrug resistance in tumor cells. Consistent with this is that S. pombe cells harboring bfr1+ on pDB248' are resistant to actinomycin D, cerulenin, and cytochalasin B, as well as to BFA. The relative positions of the ATP-binding sequences and the clusters of transmembrane sequences within the bfr1+ protein are, however, transposed in comparison with those in P-glycoprotein; the bfr1+ protein has N-terminal ATP-binding sequence followed by transmembrane segments in each half of the molecule. The bfr1+ protein exhibited significant homology in primary and secondary structures with two recently identified multidrug resistance gene products of Saccharomyces cerevisiae, Snq2 and Sts1/Pdr5/Ydr1. The bfr1+ gene is not essential for cell growth or mating, but a delta bfr1 mutant exhibited hypersensitivity to BFA. We propose that the bfr1+ protein is another member of the ATP-binding cassette superfamily and serves as an efflux pump of various antibiotics.

Uso1 protein contains a coiled-coil rod region essential for protein transport from the ER to the Golgi apparatus in Saccharomyces cerevisiae.

We have previously shown that the Saccharomyces cerevisiae USO1 gene required in the protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus encodes a 200-kDa protein (1,790 amino acids) which is present in a nonglobular high molecular mass complex. Antibodies against an N-terminal portion of Uso1 protein recognized a 100-kDa protein in Western blot of the temperature-sensitive uso1-1 mutant cell lysate. The nucleotide sequence of uso1-1 indicated the 951st codon was UAG (amber) in place of CAG (glutamine) in USO1. Deletion study of USO1 gene indicated that such truncated Uso1 polypeptides are sufficiently functional at 25 degrees C but not at 37 degrees C. Mutant Uso1-1 protein displayed an apparent molecular mass of 400-500 kDa in gel filtration while it cosedimented with a globular 6S marker protein, horseradish peroxidase (44 kDa), in sucrose density gradient centrifugation. These results indicated that truncated Uso1-1 protein is still present in a nonglobular high molecular mass complex, similar to the wild-type Uso1 protein.

[A case of vertebral metastasis revealed by incomplete spinal analgesia for cesarean section].

A 36-year-old woman was scheduled for Cesarean section under spinal anesthesia. She was a carrier of hepatitis-B-virus and diabetic. She was complaining of low back pain. Spinal anesthesia was performed in the left lateral decubitus position. Because lumbar puncture in the midline was difficult, left paramedian approach was tried. Then she began to complain of right leg pain. Another attempt was made at other site, but her pain was not relieved. After confirming drop of blood-tinged cerebrospinal fluid, 0.3% dibucaine 2.0 ml was injected. Sensory anesthesia was assessed by pin-prick, but anesthesia was not effective. Then epidural catheter was inserted at Th12-L1 using median approach. She received 1.0% lidocaine 15 ml. However, sensory anesthesia was insufficient (Th4-Th12). Therefore O2-N2O was administered in addition to regional anesthesia. After the delivery, she still complained of low back pain. Later examination revealed metastatic bone tumor of L2 from hepatoma. This case suggests that in a patient with such incomplete spinal or epidural anesthesia and neurological finding, vertebral metastatic tumor should be ruled out.