Role of prolipoprotein diacylglyceryl transferase (Lgt) and lipoprotein-specific signal peptidase II (LspA) in localization and physiological function of lipoprotein MsmE in Streptococcus mutans.

INTRODUCTION: To clarify the role that prolipoprotein diacylglyceryl transferase (Lgt) and lipoprotein-specific signal peptidase II (LspA) play in the physiological function of MsmE, we constructed lgt-deficient and lspA-deficient mutants of Streptococcus mutans 109c and examined the potential role of Lgt and LspA in membrane anchoring and growth in a melibiose medium of S. mutans. METHODS: The lgt-, lspA-, and msmE-deficient mutants of S. mutans 109c were constructed by double-crossover recombination of their respective genes. Localization of MsmE was demonstrated by Western blot analysis with an MsmE antiserum. The growth of S. mutans cells was examined in a Trypton medium containing melibiose or glucose. RESULTS: In the S. mutans lgt mutant, localization of the surface lipoprotein MsmE changed with the culture supernatant. The growth of the S. mutans lgt and lspA mutants was remarkably reduced in the melibiose medium; however, growth was recovered in the strains complemented with the lgt or the lspA gene. Therefore, lipid-modification by Lgt and subsequent signal peptide cleavage by LspA were crucial for membrane anchoring and the physiological function of MsmE in S. mutans. CONCLUSION: These results demonstrate that MsmE is required for melibiose metabolism in S. mutans and that modification by Lgt and LspA are important processes for the physiological function of MsmE.

Pulmonary exposure to diesel exhaust particle components enhances circulatory chemokines during lung inflammation.

This study examines the effects of DEP components on circulatory CC and CXC chemokines, potent activators and chemoattractants for macrophage and leukocyte subpopulations, in a murine model of lung inflammation. ICR mice were divided into six experimental groups which received intratracheal inoculation of vehicle, LPS alone (2.5 mg/kg), organic chemicals in DEP (DEP-OC: 4 mg/kg) extracted with dichloromethane, residual carbonaceous nuclei after the extraction (washed DEP: 4 mg/kg), DEP-OC + LPS, or washed DEP + LPS. Intratracheal instillation of each DEP component alone did not significantly change the circulatory level of macrophage inflammatory protein (MIP)-1alpha, MIP-2, and macrophage chemoattractant protein-1 (MCP-1) 24 h after the exposure as compared with vehicle instilled alone. In the LPS group, MCP-1, but not MIP-1alpha or MIP-2, was significantly greater than in the vehicle group. The combined administration of LPS and washed DEP caused a further three to five-fold increase in MIP-1alpha, MIP-2, and MCP-1 proteins in the serum as compared with LPS administered alone. No significant difference between the LPS + DEP-OC group and the LPS group was observed. These results indicate that pulmonary exposure to washed DEP enhances circulatory level of chemokines during lung inflammation. The enhancement may be important in the aggravations of systemic inflammatory responses and ischemic cardiovascular conditions associated with air pollution.

Nucleases from Prevotella intermedia can degrade neutrophil extracellular traps.

Periodontitis is an inflammatory disease caused by periodontal bacteria in subgingival plaque. These bacteria are able to colonize the periodontal region by evading the host immune response. Neutrophils, the host's first line of defense against infection, use various strategies to kill invading pathogens, including neutrophil extracellular traps (NETs). These are extracellular net-like fibers comprising DNA and antimicrobial components such as histones, LL-37, defensins, myeloperoxidase, and neutrophil elastase from neutrophils that disarm and kill bacteria extracellularly. Bacterial nuclease degrades the NETs to escape NET killing. It has now been shown that extracellular nucleases enable bacteria to evade this host antimicrobial mechanism, leading to increased pathogenicity. Here, we compared the DNA degradation activity of major Gram-negative periodontopathogenic bacteria, Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans. We found that Pr. intermedia showed the highest DNA degradation activity. A genome search of Pr. intermedia revealed the presence of two genes, nucA and nucD, putatively encoding secreted nucleases, although their enzymatic and biological activities are unknown. We cloned nucA- and nucD-encoding nucleases from Pr. intermedia ATCC 25611 and characterized their gene products. Recombinant NucA and NucD digested DNA and RNA, which required both Mg2+ and Ca2+ for optimal activity. In addition, NucA and NucD were able to degrade the DNA matrix comprising NETs.

Pressure-induced nano-crystallization of silicate garnets from glass.

Transparent ceramics are important for scientific and industrial applications because of the superior optical and mechanical properties. It has been suggested that optical transparency and mechanical strength are substantially enhanced if transparent ceramics with nano-crystals are available. However, synthesis of the highly transparent nano-crystalline ceramics has been difficult using conventional sintering techniques at relatively low pressures. Here we show direct conversion from bulk glass starting material in mutianvil high-pressure apparatus leads to pore-free nano-polycrystalline silicate garnet at pressures above ∼10 GPa in a limited temperature range around 1,400 °C. The synthesized nano-polycrystalline garnet is optically as transparent as the single crystal for almost the entire visible light range and harder than the single crystal by ∼30%. The ultrahigh-pressure conversion technique should provide novel functional ceramics having various crystal structures, including those of high-pressure phases, as well as ideal specimens for some mineral physics applications.

Purification and characterization of alkaline phosphatase containing phosphotyrosyl phosphatase activity from the bacterium Prevotella intermedia.

A novel alkaline phosphatase, designated PiALP, has been purified and characterized from Prevotella intermedia ATCC 25611, an anaerobe implicated in progressive periodontal disease. The enzyme was a homodimer of apparently identical subunits of Mr 54 kDa. Thiol-reducing agents completely inhibited the purified enzyme. The enzyme was highly stable even at 80 degrees C. It exhibited substantial activity against tyrosine-phosphate-containing Raytide. The phosphatase activity was sensitive to orthovanadate and Zn2+ but highly resistant to okadaic acid. The amino acid sequence of peptides derived from PiALP showed a high degree of identity (65%) with alkaline phosphatases from Zymomonas mobilis and Synechococcus. The present results imply that PiALP might represent a new family of alkaline phosphotyrosyl phosphatases which has not been described previously.

Generation of reactive oxygen species during interaction of diesel exhaust particle components with NADPH-cytochrome P450 reductase and involvement of the bioactivation in the DNA damage.

Since the toxicity of diesel exhaust particles (DEP) after intratracheal injection, was suppressed by pretreatment with superoxide dismutase (SOD) modified with polyethylene glycol (Sagai et al. Free Rad. Biol. Med. 14: 37-47; 1993), the possibility that superoxide could be enzymatically and continuously generated from diesel exhaust particles (DEP), was examined. Nicotinamide-adenine dinucleotide phosphate, reduced (NADPH) oxidation was stimulated during interaction of a methanol extract of DEP with the Triton N-101 treated microsomal preparation of mouse lung whereas the cytosolic fraction was less active, suggesting that DEP contains substrates for NADPH-cytochrome P450 reductase (EC 1.6.2.4, P450 reductase) rather than DT-diaphorase. When purified P450 reductase was used as the enzyme source, the turnover value was enhanced approximately 260-fold. Quinones appeared to be served as substrate for P450 reductase because reaction was inhibited by addition of glutathione (GSH) to form those GSH adduct or pretreatment with NaBH4 to reduce those to the hydroxy compounds although a possibility of nitroarenes as the alternative substrates cannot be excluded. A methanol extract of DEP (37.5 micrograms) caused a significant formation of superoxide (3240 nmol/min/mg protein) in the presence of P450 reductase. Electron spin resonance (ESR) experiments revealed that hydroxyl radical was formed as well. The reactive species generated by DEP in the presence of P450 reductase caused DNA scission which was reduced in the presence of superoxide dismutase (SOD), catalase, or hydroxyl radical scavenging agents. Taken together, these results indicate that DEP components, probably quinoid or nitroaromatic structures, that appear to promote DNA damage through the redox cycling based generation of superoxide.

Intraoperative radiotherapy for esophageal carcinoma--significance of IORT dose for the incidence of fatal tracheal complication.

PURPOSE: The feasibility of intraoperative radiotherapy (IORT) combined with modified regional lymphatic dissection (plus esophagectomy) for advanced esophageal carcinoma was tested. The quality of life in the patients was expected to improve by modified surgery, securing a good local control by additional IORT. METHODS AND MATERIALS: Total esophagectomy plus modified three-regional lymphatic dissection with upper mediastinal IORT followed by postoperative external beam irradiation was systematically given to 62 patients between August 1989 and June 1992. Sixty-five percent of the patients were age over 60, and 76% (47/62) of the patients were Stage III or IV by pTNM. Several techniques for the IORT were developed and used throughout this period, including a temporary collapse of the right lung by unilateral tracheal incubation (for the insertion of IORT applicator) and an in vivo dosimetry to know the appropriate range (energy) of electron beam. The method of surgical treatment, the dose of external beam irradiation were kept standardized, and only the dose of IORT was randomized either to 20 or 25 Gy. IORT-related complications and the pattern of failures were carefully monitored. RESULTS: (a) Most prominent IORT-related complication was the late tracheal damage, which occurred 6 of 44 patients who were at risk for more than a year. (b) The incidence of IORT-induced tracheal damage was sharply dependent on the dose of IORT; 6 out of 21 patients who received single dose of 25 Gy, and none out of 33 who were given 20 Gy or less. (c) 2-year cause-specific survival and actuarial 2-year survival were 75.0 +/- 14.5% and 62.5 +/- 13.2%, respectively. No loco-regional recurrence has been detected at the time of analysis. CONCLUSION: IORT in combination with modified total esophagectomy is an effective and safe method to obtain a local control in advanced esophageal carcinomas, if the dose of IORT does not exceed 20 Gy.

Reduction of radiation-induced xerostomia in nasopharyngeal carcinoma using CT simulation with laser patient marking and three-field irradiation technique.

PURPOSE: Tumor control and reduction of postirradiation xerostomia in patients with nasopharyngeal carcinoma (NPC) using the three-field irradiation technique based on the CT-based simulation with laser patient marking was investigated. METHODS AND MATERIALS: Seventy-eight patients with NPC were consecutively treated between 1983 and 1993. In 33 patients treated before 1987, target volume was determined using a conventional x-ray simulator with a reference of CT images, and the primary site was treated by the conventional parallel-opposed two-field technique (Group I). In 45 patients treated from 1987, target volume was determined using a CT simulator slice by slice, the treatment field was projected onto the patient's skin by a laser beam projector mounted on a C-arm, and the primary site was irradiated by a three-fields (anterior and bilateral) technique (Group II). In Group II, the shape of each field was determined using a beam's eye view to reduce the dose to the bilateral parotid glands. The three-field technique reduced the dose to the superficial lobe of parotid gland to about two-thirds of the dose given by the two-field technique. Radiation-induced xerostomia was evaluated by clinical symptoms and radioisotope sialography. RESULTS: The 5-year survival rate and disease-free survival rate were 46.6 and 31.2% in Group I, and 46.8 and 46.5% in Group II. A large variation in the volume of parotid glands were demonstrated, ranging from 9 cm3 to 61 cm3 among patients treated with CT simulation. Forty percent of the patients in Group II showed no or mild xerostomia, whereas all of the patients in Group I showed moderate to severe xerostomia (p < 0.01). The radioisotope sialography study showed that the mean secretion ratio by acid stimulation was improved from 3.8% in the Group I to 15.2% in the Group II (p < 0.01). CONCLUSIONS: CT simulation was useful to determine the size and shape of each field to reduce the dose to the parotid gland, of which size varies largely among individual patients. The three-field technique based on CT simulation with laser patient markings is suggested to result in superior complication-free survival in terms of salivary dysfunction than did the conventional two-field technique with x-ray simulatior for NPC.

Ipsilateral irradiation for carcinomas of tonsillar region and soft palate based on computed tomographic simulation.

PURPOSE: To reduce xerostomia in selected patients with carcinomas of the tonsillar region and soft palate. METHODS AND MATERIALS: We evaluated the treatment results of 32 patients with tonsillar region and soft palate carcinoma treated by radical radiotherapy between May 1989 and December 1996. They have a unilateral tumor that did not cross midline and have no contralateral neck lymphnode metastasis and treated with an ipsilateral technique (an anterior oblique and a posterior oblique field). All patients were planned with computed tomographic (CT) simulation and given 65 Gy in 26 fractions in 6.5 weeks with or without 5-15 Gy boost irradiation. The median follow-up was 44 months (4-86 months). RESULTS: Five-year overall, cause-specific survival, local control, and regional control rate was 64, 79, 74 and 81%. No failure at the contralateral neck occurred. Moderate or severe symptomatic xerostomia was seen in 3 (9%) patients and ostero-radionecrosis requiring surgery occurred in one (3.3%) of 32 patients. CONCLUSION: It is suggested that the ipsilateral technique is indicated in patients who had an unilateral tonsillar region or soft palate carcinoma that did not cross midline and have no contralateral neck lymphnode metastasis.

High-dose-rate intracavitary irradiation using linear source arrangement for stage II and III squamous cell carcinoma of the uterine cervix.

PURPOSE: The purpose of this article is to evaluate fractionated high-dose-rate (HDR) intracavitary irradiation using linear source arrangement (LSA) for patients with squamous cell carcinoma of the uterine cervix. MATERIALS AND METHODS: The subjects consisted of 217 patients (71 patients with stage II and 146 with stage III disease) who received external beam therapy (EBT) followed by fractionated HDR intracavitary irradiation using LSA between January 1980 and June 1990. In EBT, 40 Gy in 20 fractions (40 Gy/20 Fr) or 39.6 Gy/22 Fr was delivered to the whole pelvis and an additional 10 Gy/5 Fr or 10.8 Gy/6 Fr was delivered to the parametrium. The intracavitary irradiation dose was 30 Gy/6 Fr or 35 Gy/7 Fr with a daily fraction size of 5 Gy and two fractions per week. During the intracavitary treatment, most patients were treated on an out-patient basis. RESULTS: Cause-specific 5-year survival rates were 77% for stage II and 50% for stage III. Pelvic failure rates were 13% for stage II and 36% for stage III. In multivariate analyses, improved cause-specific survival was significantly associated with stage II (P = 0.0003), higher pretreatment serum hemoglobin level (P = 0.0015), higher pretreatment serum total protein level (P = 0.0029), and shorter total treatment time (P = 0.0024). The rate of severe (grade 3 or 4) late complication was 2% for the rectum, 1% for the small intestine or sigmoid colon and 1% for the bladder. CONCLUSIONS: Fractionated HDR intracavitary irradiation using LSA is an effective treatment for patients with uterine cervical cancer without need for hospitalization.

Concurrent chemotherapy and thoracic radiation therapy for limited-stage small cell lung cancer.

We conducted a phase II study of therapy for limited-stage small cell lung cancer (LD-SCLC). The chemotherapy regimen consisted of a three-week cycle of cisplatin (80 mg/m(2), given intravenously on day 1) and etoposide (100 mg/m(2), given intravenously on days 1, 3 and 5), given three to four times. Fifty Cy thoracic irradiation was administered in standard fractions simultaneously without a treatment break. A total of 19 patients with SCLC were entered into the study, and 18 were eligible. This concurrent treatment produced 39% complete-response and 89% overall-response rates in the eligible patients. The median response duration was 36 weeks, and the median survival time was 67 weeks. A local relapse within the irradiation field was observed in 28% of the eligible patients. Brain metastasis as the first relapse was seen in 33% of the eligible patients. Myelosuppression represented by grade 3 and 4 leukopenia was experienced in 79% of the entered patients. We conclude that the concurrent modality with cisplatin and etoposide (PE) chemotherapy and early thoracic radiation therapy without split is a feasible and beneficial therapy.

Generation of methoxide from methane and oxygen under mild conditions over an Sm2O3 catalyst investigated by in situ IR spectroscopy.

The generation of methoxide over an Sm2O3 catalyst was clearly observed under a CH4-O2 atmosphere at 323 K by in situ Fourier transform IR spectroscopy.

The association of intracranial aneurysms and arteriovenous malformation of the brain. Case report.

A case of intracranial multiple aneurysms associated with an arteriovenous malformation (AVM) is described. Three aneurysms were found arising from an enlarged anterior cerebral artery feeding an AVM. In spite of the fact that two of these aneurysms received no surgical treatment, they disappeared almost completely several months after excision of the AVM. Seventy-three previously reported cases of cerebral aneurysms associated with AVM's are reviewed, and the effect of hemodynamic stresses on the development of these aneurysms is summarized.

Inhibition of constitutive nitric oxide synthase by benexate.

We evaluated the inhibitory action of benexate (benzyl 2-[trans-4 -(guanidinomethyl) cyclohexylcarbonyloxy] benzoate hydrochloride beta-cyclodextrin clathrate), an anti-ulcer agent, on the formation of nitric oxide by stomach and brain enzyme preparations and on the purified neuronal nitric oxide synthase (NOS). Benexate markedly inhibited NOS activities of both stomach and brain preparations, with IC50 values of 68 and 29 microM, respectively. The results of double-reciprocal analysis suggested that the inhibition was competitive with an arginine substrate. Experiments with purified NOS revealed that benexate suppressed not only citrulline formation but also the oxidation of NADPH and the production of hydrogen peroxide by the enzyme. Taken together, it is indicated that benexate is an inhibitor for NOS in spite of the fact the drug elicits an increase in blood flow in a gastric mucosa.

Scintigraphic evaluation of long-term salivary function after bilateral whole parotid gland irradiation in radiotherapy for head and neck tumour.

In 25 patients with head and neck tumour, in whom bilateral whole parotid glands were totally included in the irradiation volume, parotid gland function was estimated and compared with that of 58 non-irradiated parotid glands in 29 patients, by symptomatic grading and radioisotope (RI) sialography. Sequential follow-up by RI sialography has suggested that if the total dose to the parotid gland is less than 52 Gy or if the time-dose fractionation (TDF) value is less than 80, then partial recovery of salivary function can be expected 1 or 2 years after irradiation, even if patients show severe xerostomia during the first 6 months. Long-term recovery rarely occurs in parotid glands which have been irradiated at doses exceeding 55 Gy or with TDF values of 88 or higher. The 50% complication probability (ED50) was less than 33 Gy for subacute xerostomia during the first 6 months and 52.5 Gy for chronic xerostomia after 12 months, respectively. Although a prospective study will be required for confirmation, differences between the dose-response curves of xerostomia at subacute and chronic phases suggest a basis in different biological mechanisms.

The background adaptation of the flatfish, Paralichthys olivaceus.

The background adaptation of the flatfish, Paralichthys olivaceus, was studied by sliding beneath the fish a long strip of plastic sheet with various patterns to serve as a background. The body shade and pattern differed depending upon the pattern used. In general, the fish showed maximal pallor on a white background and darkened when lying on a black background. With a pattern of large checks, several black patches became conspicuous. The fish showed the same tendency in terms of response even when the left eye was covered. However, the fish was prevented from manifesting these changes when the right eye was covered. The results suggest that the left eye of the fish does not see the immediate background. This conclusion is supported by an anatomical study.

Early skin reaction following superficial proton irradiation.

Balb/c mice were irradiated on the leg in the area of the spread out Bragg peak (SOBP) of a 30 MeV proton beam. The proton beam was modulated to different ranges in order to determine the early skin reaction versus beam quality at different portions of the SOBP. The respective 50% moist desquamation doses of 1, 3, and 8.5 mm penetration were 25.9, 30.0, and 30.4 Gy. Irradiation of the superficial layer with the distal portion of the SOBP produced a significantly more severe reaction than did whole layer irradiation.

Simultaneous carboplatin and radiotherapy for all stages of head and neck squamous cell carcinoma.

The study investigated the toxicity and efficiency of the concomitant administration of radiotherapy and carboplatin to patients with head and neck carcinomas. Sixty-three patients with head and neck squamous cell carcinomas, other than nasopharyngeal cancer and Stage I (UICC) laryngeal cancers, were treated by external radiotherapy and four courses of carboplatin at a dose of 100 mg/m2 per week. In two patients, only three courses were possible due to renal toxicity. In the other 61 patients, toxicities were self-limiting and no patient required interruption of carboplatin administration. No patient required discontinuation of radiotherapy because of acute toxicity. Of 61 evaluable patients, a complete response (CR) was obtained in 11.5% and a partial response (PR) in 60.7% at 40 Gy. In 41 patients treated to 65 Gy (including two patients with maxillary sinus carcinoma, who were treated by debulking surgery), CR was obtained in 76.9% and CR+PR was 100% at the end of treatment. The actuarial survival rate of the 63 patients at 2 years was 69.2%, with a median follow-up period of 24.4 months. One of 12 patients who received salvage surgery after radical radiotherapy has died due to poor wound healing after the surgery. The schedule was safe, providing a weekly check of serum samples was possible. It is likely that the rate of local control and vocal cord preservation in laryngeal tumours might improve if concurrent carboplatin is used. Careful follow-up is required to determine the long-term effect of concomitant carboplatin administration.

Effect of the natural antioxidant 2"-O-glycosylisovitexin on superoxide and hydroxyl radical generation.

The inhibitory effect of 2"-O-glycosyl isovitexin (2"-O-GIV) isolated from green barley leaves on superoxide and hydroxyl radical generation was investigated using electron spin resonance spectroscopy. 2"-O-GIV exhibited dose-related inhibitory activity both towards superoxide generation and hydroxyl radical generation. 2"-O-GIV inhibited superoxide formation by 97% at a level of 25 mM and inhibited hydroxyl radical formation by 91% at a level of 500 microM. The results suggest that the inhibitory activity of 2"-O-GIV is due to trapping radicals.

Angiotensin-converting enzyme inhibition improves cardiac fatty acid metabolism in patients with congestive heart failure.

This study aimed to examine whether angiotensin-converting enzyme (ACE) inhibition improved cardiac fatty acid metabolism in patients with congestive heart failure (CHF). Myocardial 123I-beta-methyl-iodophenylpentadecanoic acid (123I-BMIPP) imaging was performed in 25 patients with CHF and in 10 control subjects. Myocardial 123I-BMIPP images were obtained 30 min and 4 h after tracer injection. The heart-to-mediastinum (H/M) ratio of 123I-BMIPP uptake and the washout rate of 123I-BMIPP from the myocardium were calculated. Patients were given enalapril for 6 months, and 123I-BMIPP imaging was repeated. H/M ratios on early and delayed images were lower in CHF patients than in normal controls (P<0.01). The washout rate of 123I-BMIPP from the myocardium was faster in CHF patients than in controls (P<0.01). As the severity of the New York Heart Association (NYHA) functional class increased, the H/M ratio decreased and the washout rate increased. The washout rate of 123I-BMIPP was inversely correlated with left ventricular fractional shortening (R=-0.62, P<0.01). ACE inhibition with enalapril increased the H/M ratio on delayed images (P<0.05) and reduced the washout rate of 123I-BMIPP (P<0.05) in CHF patients. These data suggest that: (1) angiotensin II-mediated intracellular signalling activation may be a possible mechanism for the decreased myocardial uptake and enhanced washout of 123I-BMIPP in heart failure patients; and (2) the improvement in fatty acid metabolism by ACE inhibition may represent a new mechanism for the beneficial effect of this therapy in heart failure.