Backscattered electron image of osmium-impregnated/macerated tissues as a novel technique for identifying the cis-face of the Golgi apparatus by high-resolution scanning electron microscopy.

The osmium maceration method with scanning electron microscopy (SEM) enabled to demonstrate directly the three-dimensional (3D) structure of membranous cell organelles. However, the polarity of the Golgi apparatus (that is, the cis-trans axis) can hardly be determined by SEM alone, because there is no appropriate immunocytochemical method for specific labelling of its cis- or trans-faces. In the present study, we used the osmium impregnation method, which forms deposits of reduced osmium exclusively in the cis-Golgi elements, for preparation of specimens for SEM. The newly developed procedure combining osmium impregnation with subsequent osmium maceration specifically visualised the cis-elements of the Golgi apparatus, with osmium deposits that were clearly detected by backscattered electron-mode SEM. Prolonged osmication by osmium impregnation (2% OsO4 solution at 40°C for 40 h) and osmium maceration (0.1% OsO4 solution at 20°C for 24 h) did not significantly impair the 3D ultrastructure of the membranous cell organelles, including the Golgi apparatus. This novel preparation method enabled us to determine the polarity of the Golgi apparatus with enough information about the surrounding 3D ultrastructure by SEM, and will contribute to our understanding of the global organisation of the entire Golgi apparatus in various differentiated cells.

Hyperbaric oxygen therapy for the placement of dental implants in irradiated patients: systematic review and meta-analysis.

This study was a systematic review with meta-analysis to evaluate the influence of hyperbaric oxygen therapy (HBOT) on the survival of dental implants placed in patients who had had radiotherapy for cancer of the head and neck. A systematic literature search was conducted using the PubMed/Medline, Science Direct, Embase and the Cochrane Library, between January 1985 and July 2018. The study observed the PRISMA (Preferred Reporting Items for Systematic review and Meta-Analysis) declaration and norms, and the systematic review was duly recorded in the PROSPERO (International prospective register of systematic reviews) database. Inclusion and exclusion criteria were applied, and all articles were selected on the basis of PICO questions. The process of eligibility and quality evaluation yielded three studies for statistical analysis. Based on the survival rates, there was no evidence that the risk of an implant failing was different between the patients who received HBOT and those who did not. Moreover, the risk of an implant failing did not depend on the anatomical site. HBOT exerted no beneficial influence on the survival rates of implants placed in irradiated patients, and the risk of an implant failing did not depend on its location.

Do screws and screw holes affect osteolysis in cementless cups using highly crosslinked polyethylene? A 7 to 10-year follow-up case-control study.

BACKGROUND: The use of screws and the presence of screw holes may cause acetabular osteolysis and implant loosening in cementless total hip arthroplasty (THA) using conventional polyethylene. In contrast, this issue is not fully understood using highly crosslinked polyethylene (HXLPE), particularly in large comparative study. Therefore, we performed a case-control study to assess the influence of screw usage and screw holes on: (1) implant fixation and osteolysis and (2) polyethylene steady-state wear rate, using cases with HXLPE liners followed up for 7-10 years postoperatively. HYPOTHESIS: The screw usage and screw holes adversely affect the implant fixation and incidence of wear-related osteolysis in THA with HXLPE. PATIENTS AND METHODS: We reviewed 209 primary cementless THAs performed with 26-mm cobalt-chromium heads on HXLPE liners. To compare the effects of the use of screws and the presence of screw holes, the following groups were established: (1) with-screw (n=140); (2) without-screw (n=69); (3) no-hole (n=27) and (4) group in which a cup with screw holes, but no screw was used (n=42). Two adjunct groups (no-hole cups excluded) were established to compare the differences in the two types of HXLPE: (5) remelted group (n=100) and (6) annealed group (n=82). Implant stability and osteolysis were evaluated by plain radiography and computed tomography. The wear rate from 1 year to the final evaluation was measured using plain X-rays and PolyWare Digital software. RESULTS: All cups and stems achieved bony fixation. On CT-scan, no acetabular osteolysis was found, but there were 3 cases with a small area of femoral osteolysis. The mean steady-state wear rate of each group was (1) 0.031±0.022, (2) 0.033±0.035, (3) 0.031±0.024, (4) 0.029±0.018, (5) 0.030±0.018 and (6) 0.034±0.023mm/year, respectively. A comparison of the effects of screw usage or screw holes found no significant between-group differences in the implant stability, prevalence of osteolysis [no acetabular osteolysis and 3/209 at femoral side (1.4%)] and steady-state wear rate. DISCUSSION: This study suggests that there are no adverse effects on the results of THA with HXLPE from the use of cups with screw holes and the use of screws for cup fixation. LEVEL OF EVIDENCE: Level III retrospective case-control study.

Intermittent nature of solar wind turbulence near the Earth's bow shock: phase coherence and non-Gaussianity.

The link between phase coherence and non-Gaussian statistics is investigated using magnetic field data observed in the solar wind turbulence near the Earth's bow shock. The phase coherence index Cphi, which characterizes the degree of phase correlation (i.e., nonlinear wave-wave interactions) among scales, displays a behavior similar to kurtosis and reflects a departure from Gaussianity in the probability density functions of magnetic field fluctuations. This demonstrates that nonlinear interactions among scales are the origin of intermittency in the magnetic field turbulence.

Comparison of wear rate and osteolysis between second-generation annealed and first-generation remelted highly cross-linked polyethylene in total hip arthroplasty. A case control study at a minimum of five years.

BACKGROUND: There is no previous report that directly compared wear resistance of second-generation annealed highly cross-linked polyethylene with that of first-generation remelted highly cross-linked polyethylene. We therefore performed a retrospective study at a minimum of 5-year follow-up comparing second-generation annealed and first-generation remelted highly cross-linked polyethylene in order to: (1) assess wear rates and (2) compare the incidence of osteolysis between, (3) identify the frequency of complication related to the two types of highly cross-linked polyethylene. HYPOTHESIS: There is a difference in the linear wear rate and the incidence of osteolysis between the two types of highly cross-linked polyethylene in total hip arthroplasty. MATERIALS AND METHODS: In a single centre study, we reviewed 123 primary cementless total hip arthroplasties between 2010 and 2011 that were performed with 32mm alumina ceramic on second-generation annealed (X3) or first-generation remelted (Longevity) highly cross-linked polyethylene liner. There was no specific reason for the choice of the type of highly cross-linked polyethylene. There were no significant differences between the two groups in respect of gender, diagnosis, body mass index, pre- and post-operative functional and activity score, cup size, and cup orientation except the younger age in the X3 group. The mean wear rate and the incidence of osteolysis were evaluated at the latest follow-up. RESULTS: One hundred nine cases followed over 5 years post-operatively (88.6% in all consecutive cases) were evaluated. X3 and Longevity were used in 54 and 55 cases, respectively. The mean follow-up was 5.3 years in both groups. The mean linear wear rate of X3 and Longevity group was 0.045±0.023mm/year and 0.076±0.031mm/year, respectively (P<0.001). No osteolysis was found on plain X-rays in both groups and no specific complication was related to these highly cross-linked components. DISCUSSION: Excellent wear resistance of both types of highly cross-linked polyethylene liner was revealed in our study. The difference of wear rate between two materials should be monitored in a longer follow-up. LEVEL OF EVIDENCE: Level III retrospective case control study.

Comparison of wear rate and osteolysis between annealed and remelted highly cross-linked polyethylene in total hip arthroplasty. A case control study at 7 to 10 years follow-up.

BACKGROUND: Low polyethylene wear rate and low incidence of osteolysis after total hip arthroplasty using annealed and remelted highly cross-linked polyethylene have been reported. However, there is no previous report that directly compared both types of highly cross-linked polyethylene. We therefore performed a retrospective study on a series of highly cross-linked polyethylene, in order to: (1) compare wear rates and the incidence of osteolysis between annealed and remelted highly cross-linked polyethylene at 7-10 years; (2) identify the frequency of complication related to annealed and remelted highly cross-linked polyethylene. HYPOTHESIS: There is no difference in the linear wear rate and the incidence of osteolysis between the annealed and remelted highly cross-linked polyethylene in total hip arthroplasty. PATIENTS AND METHODS: Two hundred and sixteen cases of cementless total hip arthroplasties with annealed or remelted highly cross-linked polyethylene, which were performed between January 2003 and December 2006 in one institution, were followed for 7-10 years and received computed tomography scan, in addition to radiography at the latest follow-up. Annealed and remelted highly cross-linked polyethylene was used in 91 cases and 125 cases, respectively. A 26-mm cobalt-chromium head was used in all cases. Penetration rates from 1 year to the last evaluation were used to estimate the yearly linear wear rate. Existence of osteolysis was evaluated by plain radiography and computed tomography. RESULTS: There were no significant differences in patients' background between the two groups. The linear wear rate of annealed and remelted group was 0.031±0.022mm/year and 0.032±0.020mm/year, respectively (P=0.91). Two cases of small femoral osteolysis were found in the annealed group. Any complication related to highly cross-linked polyethylene was not found in both groups. DISCUSSION: There was no significant difference in the linear wear rate and the incidence of osteolysis between the annealed and remelted group at postoperative 7 to 10 years. Excellent results of both types of highly cross-linked polyethylene were revealed by this study. LEVEL OF EVIDENCE: Level III retrospective case control study.

Association of a myristoylated protein with a biological membrane and its increased phosphorylation by protein kinase C.

A hydrophilic enzyme, lysozyme, was myristoylated in vitro by the N-hydroxysuccinimide ester of myristic acid, and the monomyristoylated lysozyme was isolated by CM-cellulose cation-exchange column chromatography. The monomyristoylated lysozyme associated with phospholipid vesicles, whereas the association of native lysozyme was negligible. The membrane-associated monomyristoylated lysozyme was phosphorylated with partially purified rat brain Ca2+- and phospholipid-dependent protein kinase (protein kinase C) in the presence of Ca2+, phosphatidylserine and phorbolmyristate acetate. Thus, the myristoylated lysozyme became a substrate of protein kinase C through its hydrophobic association with the membrane. The present results suggest that the myristoylation of cytoplasmic proteins may have an important role in signal transduction.

Biochemistry of chitinases.

Chitinases are found in many organisms, and their properties seem to be closely related to their biological function. In this chapter, the physicochemical properties of chitinases such as molecular size are compared among organisms, and the optimum and stability conditions for chitinase activity are described. Furthermore, considering their classification based on amino acid sequence, kinetic behaviors are discussed together with their biological functions. In particular, hydrolytic mechanisms such as inversion and retention of the substrate are discussed in relation to allosamidin inhibition.

A genomic clone for a chitinase gene from the silkworm, Bombyx mori: structural organization identifies functional motifs.

A genomic clone containing the chitinase gene was isolated and characterized from the silkworm, Bombyx mori, using the polymerase chain reaction technique directed by primers designed from the chitinase cDNA of the insect previously cloned [Insect Biochem. Molec. Biol. 28 (1998) 163]. Results from nucleotide sequence analysis of the gene and its PCR amplification suggest that the B. mori genome probably has only one copy of chitinase gene. The gene is organized into 10 coding regions, exons, interrupted by noncoding regions, introns. The motifs encoded and exon organization of the gene are almost identical to the related gene from Manduca sexta. The results suggest that the domain organization of chitinase genes may be conserved among insect cuticular chitinase genes and that they are more complex than their counterparts in plants. The possibilities of intron splicing from the primary transcripts are also discussed.

Purification and characterization of Bombyx mori chitinases.

Two isozymes of chitinase (EC 3.2.1.14) were purified from the fifth-instar larvae of Bombyx mori by chromatography on DEAE-Cellulofine A-500, hydroxylapatite, Butyl-Toyopearl 650M, and Fractogel EMD DEAE 650 (M). These two isozymes were glycoproteins with different apparent molecular masses of 65 and 88 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pHs of the 65 and 88 kDa chitinases were 5.5 and 6.5, respectively, towards a short substrate, N-acetylchitopentaose (GlcNAc5), whereas their high activities were observed in a wide pH range between 4 and 10 towards a longer substrate, glycolchitin. Steady-state kinetic analysis of these chitinases was performed using a series of N-acetylchitooligosaccharides (GlcNAcn, n = 2-6) and glycolchitin as the substrates. Kinetic parameters for both chitinases could be obtained in the hydrolysis of glycolchitin, but not in that of N-acetylchitooligosaccharides because of strong substrate inhibition. Both chitinases similarly hydrolysed N-acetylchitooligosaccharides except for GlcNAc2 as follows: GlcNAc3 to GlcNAc plus GlcNAc2, GlcNAc4 to two molecules of GlcNAc2, GlcNAc5 to GlcNAc2 plus GlcNAc3, and GlcNAc6 to GlcNAc2 plus GlcNAc4 as well as two molecules of GlcNAc3. These results suggest that these chitinases are endo-type hydrolases, and preferred the longer-chain N-acetylchitooligosaccharides. With respect to activity, the 65 kDa chitinase was 1.7-fold more active than the 88 kDa chitinase with regard to the initial velocity in the reaction of 0.1 mM N-acetylchitooligosaccharides (GlcNAcn, n = 3-6), whereas in the overall reaction of glycolchitin (kcat/K(m)), the 88 kDa chitinase was four times more active than the 65 kDa chitinase. Regarding the affinity (1/K(m)) to glycolchitin, the affinity of the 88 kDa chitinase was 5.8-fold higher than that of the 65 kDa chitinase. The protein amino acid and gene nucleotide sequences were partly determined. Both N-terminal amino acid sequences of the 65 and 88 kDa chitinases were identical as ADSRARIVXYFSNWAVYRPG. The partial amino acid (113 amino acids) and nucleotide sequences (278 nucleotides) analysed from a mixture of 65 and 88 kDa chitinases included the two conserved regions of the family of 18 glycosyl hydrolases. All these results suggest that the B. mori chitinases are similar to Manduca sexta chitinase in primary structure and kinetic behaviour, and may be involved in the initial and intermediate stages of chitin degradation.

Activation process of pepsinogen.

The activation process of pepsinogen was analyzed by a combination of computer simulation and experiment. In order to investigate in detail the behavior of the basic schemes proposed in the previous study, further computer simulations were conducted. Some experiments were performed based on the information obtained. The changes in the UV difference spectrum in the early stage was measured by the stopped-flow technique and the conversion of pepsinogen to pepsin [EC 3.4.23.1] was followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, on the basis of the experimental results, the most reasonable scheme was selected and modified. As a result, a scheme for the activation process of pepsinogen was obtained (Scheme 8). On the basis of the above analyses, it was assumed that the first step and the third step are pH-dependent based on the change in the UV spectrum, that the second step is a nonlinear reaction containing a looped reaction with a dimeric intermediate (in this step, peptide fragments are released and pepsinogen is converted to a pepsin-like molecule), and that the third step is an equilibrium reaction involving proton binding.

Identification of residue 103 in hen egg-white lysozyme.

Since it has been uncertain whether residue 103 in hen egg-white lysozyme is aspartic acid or asparagine, we reexamined the identity of this residue. To avoid complication, the tryptic peptide T-13 (Ile 98-Arg 112) was further cleaved. The peptide containing residues Gly 102-Arg 112 was obtained by tryptic digestion of lysozyme modified at Asp 101 with diethylenetriamine. The peptide containing residues Ile 98-homoserine 105 was obtained by BrCN treatment of peptide T-13. Both Edman degradation of the former peptide and carboxypeptidase X digestion of the latter peptide identified residue 103 in hen egg-white lysozyme as asparagine.

Experimental evidence of phase coherence of magnetohydrodynamic turbulence in the solar wind: GEOTAIL satellite data.

Magnetohydrodynamic (MHD) turbulence is commonly observed in the solar wind. Nonlinear interactions among MHD waves are likely to produce finite correlation of the wave phases. For discussions of various transport processes of energetic particles, it is fundamentally important to determine whether the wave phases are randomly distributed (as assumed in the quasi-linear theory) or have a finite coherence. Using a method based on the surrogate data technique, we analysed the GEOTAIL magnetic field data to evaluate the phase coherence in MHD turbulence in the Earth's foreshock region. The results demonstrate the existence of finite phase correlation, indicating that nonlinear wave-wave interactions are in progress.

Dental extractions and radiotherapy in head and neck oncology: review of the literature.

Management of irradiated patients with cancer in the head and neck region represents a challenge for multidisciplinary teams. Radiotherapy promotes cellular and vascular decrease that results in a low response rate in the healing. Consequently, surgical procedures in irradiated tissues present high rates of complication. Osteoradionecrosis (ORN) is the most severe sequelae caused by radiotherapy. It is associated with previous extractions especially those carried out post-irradiation. The management of this side effect is difficult and can result in bone or soft tissue loss, affecting the quality of life. The literature regarding dental extractions performed before and after head and neck radiotherapy was evaluated, focusing on indications, criteria, surgical techniques and adjunctive therapies such as antibiotics and hyperbaric oxygen. Osteoradionecrosis can be minimized by oral evaluation and care prior to irradiation and healing time which allows tissue repair until the commencement of radiotherapy. In dental extractions realized after irradiation, minimal trauma, alveolectomy, primary alveolar closure and adjunctive therapies are recommended. Patients must be evaluated before radiation therapy and at that time all unrestorable teeth and/or teeth with periodontal problems must be extracted to reduce the post-radiotherapy exodontias that contribute to ORN. Once dental extractions become unavoidable after irradiation, additional care is needed.

Reversed-phase high-performance liquid chromatography of fullerenes with tetrahydrofuran-water as a mobile phase and sensitive ultraviolet or electrochemical detection.

Fullerenes (C60 and C70) were separated on a reversed-phase high-performance liquid chromatography column with a mobile phase of tetrahydrofuran-water (70:30, v/v) and detected with ultraviolet or electrochemical detection. A baseline resolution of C60 and C70 was achieved with a capacity factor values of 7.6 and 10.8 respectively and a resolution of 3.7. The detection limit for C60 was 0.1 pmol on column and was nearly the same for both detection methods.

HPLC Analysis of Anomeric Formation and Cleavage Pattern by Chitinolytic Enzyme.

The reactions of N-acetylchitooligosaccharides with chitinolytic enzyme were analyzed by HPLC using a Tosoh TSK-Gel amide-80 column with 70% acetonitrile as an eluent. We separated α and ß anomeric forms of N-acetylchitooligosaccharides, and obtain the following advantages of this HPLC method. 1. We can easily identify the reaction mechanism of chitinolytic enzymes by this method, distinguishing the inverting mechanism showing α anomer formation from the retaining mechanism showing ß anomer formation. 2. We can also estimate the cleavage patterns of N-acetylchitooligosaccharides by chitinolytic enzymes by using natural substrates.

Liquid chromatographic-atmospheric pressure chemical ionization mass spectrometric determination of anandamide and its analogs in rat brain and peripheral tissues.

A simple and selective method for the determination of anandamide (arachidonoylethanolamide), an endogenous cannabinoid receptor ligand, and its analogs with liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS) was developed. The calibration curve for standard anandamide was linear over the range 625 fmol-125 pmol per injection (r = 0.999) with a precision of 1.0% (C.V.) at 25 pmol. The detection limit attained was 200 fmol per injection at a signal-to-noise ratio of 2. Anandamide and its analogs were extracted from rat brain and peripheral tissues according to the method of Folch, and the recovery of anandamide from rat brain homogenates was 67.0-72.6%. The method was applied to their determination in rat brain and peripheral tissues.

Purification and characterization of beta-N-acetylhexosaminidase from the liver of a prawn, Penaeus japonicus.

Beta-N-Acetylhexosaminidase (EC 3.2.1.52) was purified from the liver of a prawn, Penaeus japonicus, by ammonium sulfate fractionation and chromatography with Sephadex G-100, hydroxylapatite, DEAE-Cellulofine, and Cellulofine GCL-2000-m. The purified enzyme showed a single band keeping the potential activity on both native PAGE and SDS-PAGE. The apparent molecular weight was 64,000 and 110,000 by SDS-PAGE and gel filtration, respectively. The pI was less than 3.2 by chromatofocusing. The amino-terminal amino acid sequence was NH2-Thr-Leu-Pro-Pro-Pro-Trp-Gly-Trp-Ala-?-Asp-Gln-Gly-Val-?-Val-Lys-Gly- Glu-Pro-. The optimum pH and temperature were 5.0 to 5.5 and 50 degrees C, respectively. The enzyme was stable from pH 4 to 11, and below 55 degrees C. It was 39% inhibited by 10 mM HgCl2. Steady-state kinetic analysis was done with the purified enzyme using N-acetylchitooligosaccharides (GlcNAcn, n = 2 to 6) and p-nitrophenyl N-acetylchitooligosaccharides (pNp-beta-GlcNAcn, n = 1 to 3) as the substrates. The enzyme hydrolyzed all of these substrates to release monomeric GlcNAc from the non-reducing end of the substrate. The parameters of Km and kcat at 25 degrees C and pH 5.5 were 0.137 mM and 598 s-1 for pNp-beta-GlcNAc, 0.117 mM and 298 s-1 for GlcNAc2, 0.055 mM and 96.4 s-1 for GlcNAc3, 0.044 mM and 30.1 s-1 for GlcNAc4, 0.045 mM and 14.7 s-1 for GlcNAc5, and 0.047 mM and 8.3 s-1 for GlcNAc6, respectively. These results suggest that this beta-N-acetylhexosaminidase is an exo-type hydrolytic enzyme involved in chitin degradation, and prefers the shorter substrates.

Myristoylation of protein at a distinct position allows its phosphorylation by protein kinase C.

A hydrophilic enzyme, lysozyme, was myristoylated in vitro by the N-hydroxysuccinimide ester of myristic acid and three monomyristoylated lysozymes modified at a distinct position (at Lys-13, Lys-33, Lys-97) were isolated by two-step column chromatography. The relationship between membrane binding and phosphorylation by protein kinase C of these monomyristoylated lysozymes were examined using phospholipid vesicles. These three lysozymes bound to phospholipid vesicles to the same extent, whereas the binding of nonmyristoylated native lysozyme was negligible. When native and three monomyristoylated lysozymes were reacted with protein kinase C in a phosphatidylserine (PS)-containing vesicle system, phosphorylation was observed with the myristoylated lysozymes, whereas that of native lysozyme was negligible. However, a remarkable (more than sixfold) difference in the extent of phosphorylation by protein kinase C was observed among three monomyristoylated lysozymes with a different myristoylated position. These results suggest that the membrane binding of substrate protein is not sufficient for the phosphorylation by protein kinase C and the topology of the substrate protein on the membrane play a crucial role in the recognition of substrate protein by protein kinase C. These results further indicate that protein myristoylation can modulate the topology of the membrane-bound protein.

Comparison of chitinase isozymes from yam tuber--enzymatic factor controlling the lytic activity of chitinases.

To evaluate the anti-pathogen activity of chitinases, we developed a new method for measuring the lytic activity, and investigated the correlation of the lytic activity with the enzymatic properties by using four chitinase isozymes, Chitinases E, F, H1 and G, which had been purified from yam tubers by column chromatography. Chitinases E, F and H1 had high lytic activity against the plant pathogen, Fusarium oxysporum, but Chitinase G did not. Chitinase E, which is the family 19 chitinase, was similar to Chitinases F and G in its antigenecity, but not to Chitinase H1 or H2. Chitinases H1 and H2 were recognized by the anti-Bombyx mori chitinase antibody, suggesting that Chitinases H1 and H2 are family 18 chitinases like B. mori chitinases. Chitinases E, F and H1 had two optimum pH ranges of 3-4 and 7.5-9 toward glycolchitin, but Chitinase G had only one optimum pH value of 5. Chitinases E, F and H1 had higher affinity to the polymer substrate, glycolchitin, than Chitinase G. These results suggest that the lytic activity of plant chitinases may be related to the chitin affinity and probably to the characteristic optimum pH value, or two values, but not related to its classification. The correlation of the lytic activity of a chitinase isozyme with its elicitor specificity is also discussed.