A new, direct analytical method using LC-MS/MS for fatty acid esters of 3-chloro-1,2-propanediol (3-MCPD esters) in edible oils.

A new, direct analytical method for the determination of 3-chloro-1,2-propanediol fatty acid esters (3-MCPD esters) was developed. The targeted 3-MCPD esters included five types of monoester and 25 [corrected] types of diester. Samples (oils and fats) were dissolved in a mixture of tert-butyl methyl ether and ethyl acetate (4:1), purified using two solid-phase extraction (SPE) cartridges (C(18) and silica), then analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Five monoesters and five diesters with the same fatty acid group could be separated and quantified. Pairs of 3-MCPD diesters carrying the same two different fatty acid groups, but at reversed positions (sn-1 and sn-2), could not be separated and so were expressed as a sum of both compounds. The limits of quantification (LOQs) were estimated to be between 0.02 to 0.08 mg kg(-1), depending on the types of 3-MCPD ester. Repeatability expressed as relative standard deviation (RSD(r)%) varied from 5.5% to 25.5%. The new method was shown to be applicable to various commercial edible oils and showed levels of 3-MCPD esters varying from 0.58 to 25.35 mg kg(-1). The levels of mono- and diesters ranged from 0.10 to 0.69 mg kg(-1) and from 0.06 to 16 mg kg(-1), respectively.

Purification and characterization of the plasma membrane glycosidases of Drosophila melanogaster spermatozoa.

Previous studies from our laboratory have demonstrated the presence of two integral proteins with glycosidase activity in the plasma membrane of Drosophila melanogaster spermatozoa and we have suggested that these enzymes might have a role in sperm-egg binding. In this study the glycosidases have been purified and characterized. We have evidenced the presence of three distinct enzymes, two beta-N-acetylhexosaminidase isoforms, named HEX 1 and HEX 2, and an alpha-mannosidase. The molecular size of the native enzymes estimated by gel filtration was 158 kDa for beta-hexosaminidases and 317 kDa for alpha-mannosidase. SDS-PAGE showed that HEX 1 and HEX 2 are dimers formed by subunits with different molecular sizes, whereas alpha-mannosidase consists of three subunits with different molecular weights. All the enzymes are terminally glycosylated. Characterization of the purified enzymes included their 4-methylumbelliferyl-substrate preferences, kinetic properties, inhibitor constants and thermal stability. On the basis of substrate specificity, kinetics and the results of inhibition studies, beta-hexosaminidases appear to differ from each other. HEX 1 and HEX 2 are similar to mammalian isoenzyme A and isoenzyme B, respectively. These findings represent the first report on the characterization of sperm proteins that are potentially involved in interactions with the egg in Insects.

Pathogenicity of Leucocytozoon caulleryi for specific pathogen-free laying hens.

The pathogenicity of Leucocytozoon caulleryi against specific-pathogen-free laying hens was investigated. Many large schizonts (second-generation schizonts) of L. caulleryi were seen in the ovary and oviducts of chickens. Edema and pressure atrophy of the adjacent tissues were associated with these schizonts. The eggshell-secreting portion of the uterus exhibited the most severe damage in the oviduct. This experiment reconfirms that L. caulleryi may stop egg production in laying hens, presumably as a result of damage to ovaries and oviducts.

Metachronous triple cancers of the sigmoid colon, stomach, and esophagus: report of a case.

We report herein an unusual case of metachronous triple cancers of the sigmoid colon, stomach, and esophagus. A 60-year-old man was initially admitted to our hospital for investigation of occult fecal blood. This was found to be caused by sigmoid colon cancer which was resected in July 1985 (T3, N0, M0; Stage II). A follow-up endoscopy performed in 1990 showed early gastric cancer, and a gastrectomy was performed in August 1990 (Tis, N0, M0; Stage 0). Another endoscopic examination performed as follow-up in 1993 revealed early cancer of the remnant stomach, and all the remnant stomach was surgically resected in March 1993 (Tis, N0, M0; Stage 0). He presented again in December 1996, complaining of discomfort in the chest which was found to be caused by cancer of the middle thoracic esophagus. Although surgery was considered necessary, the patient refused to undergo any further operations. Instead, radiation was administered from January 1997. An endoscopy after the completion of radiotherapy confirmed that the cancer had almost disappeared; however, it started to grow again from the beginning of 1998. He was hospitalized due to esophageal stenosis in April 1998, and died of carcinomatous cachexia in September of the same year.

Lesions of bone and bone marrow in myeloid leukosis occurring naturally in adult broiler breeders.

Lesions of bone and bone marrow in myeloid leukosis (ML) occurring naturally in adult broiler breeders were investigated pathologically. During gross examination, nodules and protrusions were commonly observed on the surface of the sternum, ribs, vertebrae, and synsacrum. The bone marrow of all the bones of the body was pale in color. Histologically, granulated myelocytes proliferated in the bone marrow of various bones and in the periosteum of the sternum, ribs, vertebrae, and synsacrum. The first proliferation of tumor cells occurred in the bone marrow of epiphysis. The myelocytes invaded through haversian and Volkmann's canals from the bone marrow to periosteal areas. Hematopoiesis was suppressed by marked proliferation of tumor cells in the bone marrow of the whole bone. Atrophy was also seen in the bones, including medullary bones of the chickens suffering from ML. Proliferation of myelocytes was seen in the bone marrow and periosteum of ossified cartilaginous rings of the trachea and larynx. Marked proliferation of myelocytes was seen in the dura mater of spinal cords, and it subsequently depressed the spinal cords. Bone formation with cartilage was seen in the periosteum of the sternum having marked proliferation of myelocytes in the bone marrow and periosteum. Ultrastructurally, tumor cells showed large nuclei and cytoplasm with large round electron-dense lysosomes. The virus particles were rarely detected in the cytoplasm of tumor cells. The polymerase chain reaction test of tumor samples showed positive for subgroup J avian leukosis virus. This study indicates that the myelocytes can invade through the compact bones to the periosteum in the sternum, ribs, vertebrae, synsarcum, and ossified cartilage of trachea and larynx having thinner compact bones. In addition, the periosteal osteogenesis with cartilage in the sternum may be reactive change against the bone atrophy because of the marked proliferation of myelocytes.

Localization of biologically uncommon D-beta-aspartate-containing alphaA-crystallin in human eye lens.

PURPOSE: Previous studies demonstrated that the Asp-151 residue of alphaA-crystallin from human eye lens is stereoinverted to the biologically uncommon D-isomer and isomerized to the beta-aspartyl residue (isoaspartate) with age. To detect the locality of the D-beta-Asp-containing peptide in aged human lens, we prepared a highly specific antibody against peptide Gly-Leu-D-beta-Asp-Ala-Thr which corresponds to positions 149-153 of human alphaA-crystallin using peptide Gly-Leu-D-beta-Asp-Ala-Thr-Gly-Leu-D-beta-Asp-Ala-Thr-Gly-Leu-D-beta- Asp-Ala-Thr (designated peptide 3R) as an immunogen. METHODS: Peptide 3R was synthesized with F-moc (9-fluorenylmethoxycarbonyl) solid phase chemistry and then the peptide was immunized in rabbits to generate antibody against peptide 3R. The antibody in rabbit serum was purified by affinity chromatography using peptide 3R and bovine alphaA-crystallin as ligands. The specificity and titer of antibody were checked by ELISA assay. We synthesized four kinds of peptide T18 (IQTGLDATHAER; corresponding to the amino acid sequences 146-157 in human alphaA-crystallin) in which Asp-151 residues were normal L-alpha-Asp, abnormal D-alpha-Asp, L-beta-Asp, and D-beta-Asp, respectively. The specificity of antibody was confirmed by ELISA using these peptides and utilized in immunohistochemistry. RESULTS: The antibody we prepared crossreacted specifically to D-beta-Asp-151-containing alphaA-crystallin. Immunohistochemical staining of human lens with the antibody demonstrated that D-beta-Asp-151-containing alphaA-crystallin was predominantly localized in the core of aged human lens. CONCLUSIONS: The peptide 3R antibody clearly recognized the presence of racemized and isomerized Asp-151 in both protein solution and lens tissue obtained from aged human lens.

Neutral and acidic glycosphingolipids in glucocorticoid-induced cataract in chick lens.

Administration of glucocorticoids induces transient cataract in 15-day-old chick embryos within 48 hr, and the opaque lens again becomes clear within the subsequent 48 hr. Oxidative stress is likely to be involved in the process of cataract formation, resulting in the appearance of numerous vacuoles around the perinuclear region. Chick lens contained low amounts of glycosphingolipids, which mainly consists of GM3, GD3, sialyl-LewisX gangliosides and glucosylceramide. Most lens gangliosides were immunohistochemically detected in lens epithelia, annular pads and developing fibers, but not in perinuclear and nuclear regions. Since cell surface gangliosides, for example GM3 and sialyl-LewisX gangliosides, are involved in cell adhesion, weak cell-to-cell interactions in the perinuclear and nuclear regions may allow vacuole formation in steroid-induced cataractogenesis.

Implication of glycolipids in lens fiber development.

Mammalian lens contains Lewis(x), sialyl-Lewis(x) and alpha-galactosyl epitopes in neolactoseries glycosphingolipids. The expression of these three epitopes is not observed in lens epithelial cells, but is immunohistochemically detected in the inner cortical fibers and the lens nucleus. In embryonic chick lens, sialyl-Lewis(x)-containing gangliosides were also detected in the transitional zone and elongating lens fibers. Thus, the Lewis(x), sialyl-Lewis(x) and alpha- galactosyl epitopes may be associated with the differentiation and maturation of lens epithelial cells to lens fibers.

Reassessment of long-term use of dense HA as dental implant: case report.

Dense hydroxyapatite (HA) is widely believed to be unsuitable for clinical use as dental implants due to its poor mechanical properties, although it has excellent biocompatibility and is chemically stable and nonresorbable in vivo. However, the case in this article is one in which the patient's dense HA implants are still stable and in good functional condition 16.5 years after he received four pieces of a one-piece dense HA implant in both sides of his lower molar regions. Furthermore, almost no radiolucency is evident along the root portions of the implant sites in the bone. These findings imply that dense HA can be clinically useful and should be reevaluated as a dental implant material.

Expression of sialylated Lewisx gangliosides in cultured lens epithelial cells from rhesus monkey.

Monkey and human lenses contain essentially the same glycosphingolipids, and Lewisx and sialylated Lewisx epitopes are expressed on the terminal structure of neolactotetraosylceramide. However, monolayer cultures of lens epithelial cells from rhesus monkey expressed gangliosides GM3, GD3 and a small amount of GM1, but not sialylated Lewisx epitopes. Eight-week-old cultures on various extracellular matrices resulted in morphological changes in lens epithelial cells. Monolayer of cells cultured on vitronectin or polylysine assembled into aggregates after 4 weeks of culture. Cells cultured on vitronectin expressed sialyl-Lewisx gangliosides and did not exhibit GD3. On collagens, fibronectin and laminin elongated cells were observed in cells cultured for 8 weeks. Thus, the interaction between cells and extracellular matrices influenced morphology and glycosphingolipid composition in lens epithelial cells.

The process of physical weakening and dissolution of the HA-coated implant in bone and soft tissue.

Hydroxyapatite (HA)-coated implants were developed to promote osseointegration of titanium implants and to overcome the mechanical drawbacks of solid HA implants. Although many clinical reports on the prognosis of HA-coated implants have reported high success rates, the risks of dissolution and weakening of the coating have been noted. We hypothesized that the chemical and mechanical stability of HA coating are affected by its microstructural characteristics. The present study investigates differences in the microstructures of available HA-coated implants, before and after implantation into the coxal bones of dogs for periods ranging from 3 weeks to 10 months and under the coxal periosteum of dogs for 10 months. The results of transmission electron microscopy and energy-dispersive x-ray analysis revealed that crystallization of super-fine HA crystals occurred in the amorphous phase of the HA coating and progressed over time. This crystallization weakens HA-coated implants by making the amorphous phase brittle, causing stress accumulation within the coating, and causing a decrease in the binding strength between the coating and the substrate. Furthermore, the HA coating dissolved in soft tissue. Dissolution started with the super-fine HA crystals in the crystallized portion that was originally part of the amorphous phase.

Differences in microstructural characteristics of dense HA and HA coating.

Two implant types of hydroxyapatite (HA) currently are available for dental implants: dense HA-cemented titanium (Ti) and HA-coated. It has been shown in previous reports that there are differences in the chemical and mechanical stabilities between the dense HA and HA coated. The differences are thought to be due to structural differences between the two ceramic types. The aim of this study was to investigate the differences in microstructural characteristics of currently available dense HA and HA coated implants before implantation and at periods of 3 weeks and 10 months after implantation in canine bone. X-ray diffractometry, infrared analysis, transmission electron microscopy, and energy dispersive X-ray analysis were used. The dense HA is composed of crystal grains, with a well crystallized structure of HA, closely bound to each other and approximately 0.4-0.6 micron in size. Implantation did not change the original sintered structure of the dense HA. The HA coating was composed of an amorphous phase with a Ca/P ratio of 1.46 and a crystal phase consisting of oxyhydroxyapatite, tricalcium phosphate, tetracalcium phosphate, and CaO, with a Ca/P ratio of 1.57. In the amorphous phase, compared to other portions in the amorphous phase, there were some layers with lower atomic density and with no significant difference in Ca/P ratio. After implantation, the crystallization of super fine crystals of approximately 4-5 nm in thickness occurred in the amorphous phase, and with time it progressed and spread from the surface to the deeper portion of the HA coating. A Ca/P ratio of 1.58 in the crystallized portion was close to the ratio (1.60) in the dense HA, suggesting that the super fine crystals were HA. This crystallization cannot significantly decrease the solubility of the amorphous phase portion and poses risks of stress accumulation within the coating and a decrease of binding strength between the HA coating and the substrate.

Localization of LewisX, sialyl-LewisX and alpha-galactosyl epitopes on glycosphingolipids in lens tissues.

Mammalian lens contains several neutral and acidic glycosphingolipids, the core structures of which are ganglio-, neolacto-, globo-, and isoglobo-series sugar chains. Old World monkey lens shows glycosphingolipid compositions similar to those of human cataractous lens, in particular the presence of Lewisxand sialyl-Lewisxepitopes and the absence of alpha-galactosyl epitope. Dog and pig lenses contain globotriaosylceramide and the sialyl-Lewisxcontaining neolactotetraosylceramide, respectively, which were found in primate lens, together with the alpha-galactosyl epitope containing neolactotetraosylceramide. Thin-layer chromatography immunostaining revealed the enrichment of some neolacto-series glycosphingolipids in the cortical and nuclear fibers, but not in lens epithelia, of dog, pig, and Japanese monkey lenses. Immunohistochemical studies confirmed the expression of Lewisx, sialyl-Lewisx, and alpha-galactosyl epitopes in the inner cortical and nuclear fibers, in association with the differentiation and maturation of lens epithelial cells to lens fibers. Glycobiological approaches thus suggested that some neolacto-series glycosphingolipids are involved in lens fiber development, in which the physiological roles of the alpha-galactosyl epitope are evolutionarily replaced by the Lewisxand sialyl-Lewisxepitopes in Old World monkeys and humans.

Comparative push-out test of dense HA implants and HA-coated implants: findings in a canine study.

Two types of hydroxyapatite (HA) implants have been developed: an HA-coated implant and a dense HA implant. For a longer in situ life span, the HA implant must remain chemically stable and possess high resistance to occlusal force. To determine which type of HA implant shows better durability, this comparative dog study was done to evaluate push-out test results of HA-coated implants and dense HA implants of approximately the same size after implantation in the mandibular and coxal bones for periods ranging from 3 weeks to 10 months. The findings revealed that for the mandibular implants, the push-out values of HA-coated implants were significantly higher than those of dense HA implants at 2 and 4 months after implantation, with significance levels of p < .001 and p < 0.05, respectively. However, there was no significant difference between the two implant types at 10 months. As for the coxal implants, no significant differences were noted for any period. Furthermore, the ratio of push-out values of the dense HA implants to those of the HA-coated implants situated in the same position bilaterally in each bone of the body for each implantation period rose with the passage of time, especially in the mandible. In the mandibular implants, the correlation coefficient of the relationship between the ratio and duration of implantation was highly significant (p < 0.001). Push-out testing caused detachment of the surface portion of the HA coating that was bound to the dense bone from the HA-coated implant at 2, 4, and 10 months after implantation. Furthermore, at 10 months the HA-coated layer in the wide areas of the implants had completely detached from the metal substrate, in contrast to the dense HA implants, which remained durable throughout the test period.

Microstructural changes in bone of HA-coated implants.

In our previous comparative push-out test of HA-coated implants and dense HA implants in dog bone, the ratio of the push-out value of the HA-coated implant to that of the dense HA implant decreased with time due to weakening of the HA coating as compared to the dense, more durable HA. The aim of this study was to investigate by histological examination of HA-coated implants in dog bone, using TEM, how this weakening of the HA coating occurs. The HA coating before implantation is composed of an amorphous glassy phase and a crystal phase scattered within the glassy phase. After implantation, the crystal phase remained almost unchanged. However, in the glassy phase, crystallization occurred and progressed with time. By 3 weeks after implantation, this crystallization already had started in the surface portion of the HA coating where it was covered by bone and also where it still touched the soft tissue. By 10 months, the crystallization had progressed to the deeper portion of the HA coating and had expanded to most of the glassy phase except for the narrow portions along the substrate-coating interface. These findings suggest that a progression of crystallization in the glassy phase causes stress accumulation within the HA coating, especially in the interface between the HA coating and the substrate, and that this stress accumulation results in a weakening of the HA-coated implant.

Bone formation on HA implants: a commentary.

The present paper reviews bone formation on dense hydroxyapatite (HA) implants. Calcification of bone matrix formed on HA is different in areas where collagen fibers are dense and scattered with matrix vesicles than in those interfacial layers containing few or no collagen fibers and matrix vesicles. Calcification of collagen-coated areas begins with crystallization within the matrix vesicles. In contrast, calcification of the interfacial layer is initiated by epitaxial crystal growth on the HA. Crystallization within the matrix vesicles near the HA and those on the HA start simultaneously. Calcification of the interfacial layer is the most important feature of HA as a biomaterial. Such calcification is never observed on titanium implants. The recent postulation that the rate of bone formation on calcium-phosphate (CP) ceramics correlates with the solubility of CP ceramics is improbable, as in vitro immersion tests have been unable to establish correlation between osteoblast differentiation and the solubility of the CP ceramics. With regard to HA-plasma spray-coated implants, future research should focus on the purity of HA and the structure of the ceramics in order to increase the in vivo chemical and mechanical durability of HA coating.

Developmental changes in carbohydrate antigens in embryonic rat lens.

The functions of glycosphingolipids, especially those containing the alpha-galactosyl epitope, were investigated during the development and differentiation of rat lens. Glycosphingolipids in embryonic lens tissue were mainly composed of neolacto-series glycosphingolipids and sialic acid-containing ganglio-series gangliosides GM3 and GD3. These glycosphingolipids and gangliosides were widely expressed on cell membranes in the lens vesicle and the elongating lens fibers. In particular, the expression of neolacto-series glycosphingolipids with the alpha-galactosyl epitope was found to be associated with the differentiation and interaction of lens fibers. Glycoproteins with the alpha-galactosyl epitope was also involved in the elongation of lens fibers. The expression of the glycoproteins was highly specific in elongating lens fibers when these were examined in head sections obtained at various embryonic stages. Thus, the alpha-galactosyl epitope on glycosphingolipids and glycoproteins appears to be associated with the differentiation and elongation of lens fibers in the rat. Evolution-related changes in the expression of carbohydrate antigens are also discussed in relation to the development and cell-to-cell interaction of lens fibers in mammals.

Gene expression in skin tumors induced in hairless mice by chronic exposure to ultraviolet B irradiation.

We investigated the expressions of c-Ha-ras, c-jun, c-fos, c-myc genes and p53 protein in the development of skin tumors induced by chronic exposure to UVB without a photosensitizer using hairless mice. When mice were exposed to UVB at a dose of 2 kJ/m2 three times a week, increased c-Ha-ras and c-myc transcripts were detected after only 5 weeks of exposure, while no tumor appeared on the exposed skin. The increase in gene expression continued until 25 weeks, when tumors, identified pathologically as mainly squamous cell carcinomas (SCC), developed in the dorsal skin. In these SCC, overexpression of c-fos mRNA was also observed along with the increases in c-Ha-ras and c-myc. A single dose of UVB (2 kJ/m2) applied to the backs of hairless mice transiently induced overexpression of the early event genes c-fos, c-jun and c-myc, but not c-Ha-ras, in the exposed area of skin. Accumulation of p53 protein was determined by Western blotting analysis or immunohistochemistry using monoclonal antibodies PAb 240 or 246, which recognize mutant or wild type, respectively. In the SCC, a mutant p53 protein accumulated in the cytoplasm and nucleus. After single-dose irradiation, the increased wild-type p53 protein was observed in the nuclei of epidermal cells. The present results suggest that overexpression of the c-fos, c-myc and c-Ha-ras genes, and the mutational changes in p53 protein might be associated with skin photocarcinogenesis. Moreover, overexpression of the c-Ha-ras and c-myc genes might be an early event in the development of UVB-induced skin tumors in mice.