Accumulation of protein-bound 4-hydroxy-2-hexenal in spinal cords from patients with sporadic amyotrophic lateral sclerosis

4-Hydroxy-2-hexenal (HHE) is a toxic, reactive aldehydic intermediate formed by nonenzymatic peroxidation of n-3 polyunsaturated fatty acids. The aim of this study was to determine the implication for HHE in the pathomechanism of amyotrophic lateral sclerosis (ALS) by immunohistochemical and enzyme-linked immunosorbent assay (ELISA) techniques using a mouse monoclonal IgG(1) antibody mAbHHE53 specific for protein-bound HHE. Immunohistochemical analysis on formalin-fixed, paraffin-embedded sections and frozen sections of spinal cords obtained at autopsy from 10 sporadic ALS patients and 10 age-matched control subjects demonstrated that protein-bound HHE immunoreactivity was seen and was prominent in the entire gray matter in the ALS cases and localized in the neurons, reactive astrocytes, microglial cells, and the surrounding neuropil, while the immunoreactivity was obscure or undetectable in the control cases. No significant protein-bound HHE immunoreactivity was seen in sections processed with omission of mAbHHE53 or in sections incubated with the antibody with an excess of the respective antigen. Competitive ELISA analysis on trypsin-digested protein extracts of fresh-frozen spinal cord samples disclosed a significant increase in protein-bound HHE level in the ALS cases compared with the control cases. Our results indicate that enhanced HHE formation occurs in the entire gray matter of sporadic ALS spinal cords and suggest that the selective vulnerability of motor neurons to HHE mediates the pathomechanism of this disease.     

Separation and characterization of adjuvant oligosaccharide oleate ester derived from product mixture of mannitol-oleic acid esterification

Nearly 30 years after intense investigations of mannide monooleates for use as vaccine adjuvants, a novel adjuvant-active saccharide oleate ester was isolated and identified from the product mixture synthesized from mannitol and oleic acid. The mixture, which contained many kinds of mannide mono- and dioleates and their derivatives, was fractionated by liquid chromatography (LC), and the fraction with the highest adjuvanticity was obtained. Gel permeation chromatography (GPC) showed that it consisted of one major compound with an average molecular weight (MW) 2850. Infrared (IR) absorption and proton nuclear magnetic resonance spectra suggested it had oligosaccharide moieties and oleate domains. These findings suggested that it was an oligosaccharide oleate ester of the average MW 2850. The molecular ratio of oleate chains per monosaccharide unit was approximately 0.8. The ester induced both IgG1 and IgG2a antibody responses in mice in a dispersed form without base oil. This ester thus appears to be one of the adjuvant-active compounds largely contributing to the excellent adjuvanticity of mannide oleate mixture broadly used as vaccine emulsifier. These results and previous findings suggest that the fundamental adjuvanticity of this 'oligo' saccharide acylate ester was in accord with the hydrophil-lipophil balance (HLB) theory, similarly to other saccharide acylate esters. It is now expected that this compound will be useful as novel vaccine adjuvant which may induce both Th1 and Th2 type immune responses with low or no toxicity, not only as an vaccine emulsifier but in an aqueous suspension form.     

High frequency of DAP-kinase gene promoter methylation in colorectal cancer specimens and its identification in serum

Death-associated protein (DAP)-kinase is frequently inactivated by promoter methylation in human cancers. To understand the involvement of the DAP-kinase gene in colorectal cancer (CRC), we investigated the methylation of the DAP-kinasegene in primary CRC to define the frequency of this epigenetic aberration and the clinicopathological significance. For this reason, methylation-specific polymerase chain reaction (MSP) was used to detect DAP-kinase gene methylation in DNA from 122 cases of CRC and 18 paired serum samples. Methylation of the DAP-kinase gene was found in 67 of 122 (55%) cases of primary CRC. Study of the serum DNA from 14 patients exhibiting methylated DAP-kinase gene revealed aberrant methylation in three patients (21%). False positives were not obtained in any of the patients who did not exhibit methylation. No association was found between the promoter methylation of the DAP-kinase gene in primary CRC and gender, localization, tumor differentiation, invasion depth, regional lymph node involvement, or tumor stage. In conclusion, methylation of the DAP-kinase gene is common in CRC. The detection of the methylation of the DAP-kinase gene has a potential clinical application as a diagnostic tumor marker for CRC.     

Usefulness of Flexible Colonoscopic Microwave Coagulation Therapy for a Colorectal Anastomotic Stricture

PURPOSE: Several methods of treatment for benign anastomotic strictures after anterior resection have been described. We describe a simple, safe, effective, and inexpensive method for treating benign colorectal anastomotic stricture by means of microwave coagulation under flexible colonoscopic visualization. METHODS: Eighteen patients with rectal or rectosigmoidal cancer underwent low anterior resection or anterior resection without colonic pouch and colorectal anastomosis by a double-stapling technique with PCEEA. Two of 18 patients (11.1 percent) developed an anastomotic stenosis. A microwave electrode was passed through the biopsy channel of the flexible colonoscope. Under flexible colonoscopic visualization, microwave irradiation was performed at four points (3, 6, 9, and 12 oclock) in the stricture site because of granulation scar to obtain an adequately coagulated area. No bougies were performed thereafter. RESULTS: The anastomotic strictures could be dilated adequately, the patients could defecate satisfactorily, and their abdominal distentions and bowel symptoms were resolved. No complications occurred. No occurrence of restricture has been observed. CONCLUSION: Flexible endoscopic, microwave coagulation therapy is a useful, simple, effective, and safe method for the treatment of benign colorectal anastomotic strictures.     

An easy and certain method to prevent dislodging of laparoscopic ports

The dislodgement of a laparoscopic port during surgery is a common problem in patients with thin abdominal walls, especially children. In this paper, we report a new technique and a simple device to fix a laparoscopic port. The device consists of an O-ring and connecting threads that are tied to the ring at two points. The connecting thread is passed though the port's cannula, and a trocar is set in the cannula beforehand. The port with the inner trocar is placed into the abdominal cavity, and the trocar is removed. The ring is brought into the abdominal cavity with a laparoscopic forceps through the cannula. After the tip of the forceps has been passed through the ring in the abdominal cavity, the thread connected to the ring is withdrawn, and the ring is fixed on the cannula by anchoring the thread on the port extracorporeally. Such a device was used to fix a total of 95 ports during 12 laparoscopic colectomies, 7 gastrectomies, 19 cholecystectomies, 2 appendectomies, and 1 resection of Meckel's diverticulum. Four children were among these cases. Not a single port slipped off, even in cases involving infants and obese patients. No complications relating to use of this device were observed. The new stabilizing device is simple, cost effective, and useful for prevention of the dislodging of various types of conventional ports, especially in the case of infants.     

Cerebral oxygenation monitor during head-up and -down tilt using near-infrared spatially resolved spectroscopy

Reflectance near-infrared spectroscopy (NIRS) has become a suitable and easily manageable method to monitor cerebral oxygenation changes in presyncopal and syncopal symptoms caused by postural changing or standing. A new clinical tissue oxygenation monitor has been recently developed which measures absolute tissue haemoglobin saturation (Tissue Oxygenation Index, TOI) utilizing spatially resolved spectroscopy (SRS). The present study examined the effects of postural changes on cerebral oxygenation as reflected in SRS-NIRS findings. Cerebral oxyhaemoglobin (O2Hb), deoxyhaemoglobin (HHb), and the TOI were recorded from both sides of the forehead in five healthy male subjects (age range, 28-40 years) during 90 degrees head-up tilt (HUT) and -6 degrees head-down tilt (HDT). Three series of measurements were carried out on separate days. O2Hb was decreased during HUT. TOI was significantly lower in HUT than in the supine position (SUP). There was no significant change in TOI during HDT. A significant session effect was observed in the left forehead TOI during SUP, but not in the right. SRS-NIRS measurements confirmed sub-clinical alterations of cortical oxygenation during HUT. NIRS data from the left side of the forehead, which may vary with cognitive or emotional activation, were more variable than those from the right side.     

Appearance of a stage-specific immunodominant glycoprotein in encysting Entamoeba invadens

The appearance of cyst-specific proteins in encysting Entamoeba invadens and their immunogenicity were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using an axenic encystation system in vitro. A rabbit antiserum against trophozoites of E. invadens reacted with a number of proteins of cysts after 1–4 days of incubation. Thus, a number of cyst proteins remained antigenically unchanged as common antigens of the two forms after transformation from trophozoites to cysts. A rabbit antiserum against cysts also reacted with the trophozoite proteins as well as the cyst proteins. The most interesting result was that the rabbit anticyst serum reacted predominantly with an 88-kDa protein of cysts after 1 day of incubation. The 88-kDa protein reacted with the anticyst serum absorbed with trophozoite proteins and was thus cyst-specific. The reactivity of the 88-kDa protein of cysts with the absorbed anticyst serum decreased as encystation proceeded. When soluble and particulate fractions prepared from cysts after 1 day of incubation were examined by electrophoresis and immunoblotting, the 88-kDa protein that had reacted with the absorbed anticyst serum was found to be present in the particulate fraction, which was rich in cell-wall fragments, and stained with periodic acid-Schiff 's reagent, indicating that it is a glycoprotein. The results indicate that encystation is accompanied by appearance of the cyst-specific 88-kDa glycoprotein, which is immunodominant and most abundantly expressed in cysts after 1 day of incubation and appears to be associated with the cyst wall. Received: 31 May 1999 / Accepted: 20 July 1999