Reconsideration of iodine in wound irrigation: the effects on Pseudomonas aeruginosa biofilm formation.

OBJECTIVE: Chronic skin wounds are usually colonised with bacteria and subsequent infection may develop. Topical antiseptics are commonly used to control bacterial colonisation. The topical antiseptic, 1% polyvinylpyrrolidone-iodine (PVP-I), that is used on chronic open skin wounds remains controversial in the clinical setting because of its cytotoxicity. Here, we tested 1% PVP-I solution against saline to determine if it reduces bacterial count on the wound surface and within the tissue that may lead to wound reduction. METHOD: Open wounds that were created on the backs of Sprague Dawley rats were inoculated with Pseudomonas aeruginosa at the wound surface. Wounds were kept covered except for wound irrigation, two days post-wounding, wounds were irrigated daily using a 10ml syringe and spray tip. RESULTS: Our results indicate that 1% PVP-I irrigation resulted in a reduced bacterial count on the wound surface and within the tissue compared with saline irrigation. The 1% PVP-I irrigation promoted wound re-epithelialisation compared with saline irrigation, but it did not reach significance. CONCLUSION: These results indicated that irrigation with 1% PVP-I was an effective way to reduce bacterial count on the wound surface, and allow the wound to progress to healing.

Involvement of high mobility group box 1 and the therapeutic effect of recombinant thrombomodulin in a mouse model of severe acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is accompanied by severe lung inflammation induced by various diseases. Despite the severity of the symptoms, therapeutic strategies have been ineffective. High mobility group box 1 (HMGB1), which was identified originally as a DNA binding protein, has been proposed as a mediator of acute lung injury. In addition to its anti-coagulant activity, recombinant thrombomodulin (rTM) possesses an ability to suppress the inflammatory response through neutralizing HMGB1. T regulatory (T(reg)) cells in the lungs are reported to modify innate immune responses during resolution of acute lung injury. In the present study, we investigated the therapeutic effect of rTM, and the contribution of T(reg) cells to this effect, in a mouse model of severe ARDS. C57BL/6 mice received sequential intratracheal administration of α-galactosylceramide (α-GalCer) and lipopolysaccharide (LPS), which resulted in the development of severe ARDS. HMGB1 levels in the lungs increased to a higher level in ARDS mice compared to those in mice treated with LPS alone. HMGB1 was expressed in the infiltrating neutrophils and macrophages in lungs. T(reg) cells were reduced significantly in the lungs of ARDS mice compared to those in mice treated with LPS alone. rTM administration prolonged the survival time and ameliorated the development of ARDS, which was associated with increased T(reg) cells and synthesis of interleukin (IL)-10 and transforming growth factor (TGF)-ß in the lungs. These results suggest that HMGB1 is involved in the development of severe ARDS and rTM shows therapeutic effects through promoting the accumulation of T(reg) cells at the inflammatory sites.

Benzylmercapturic acid is superior to hippuric acid and o-cresol as a urinary marker of occupational exposure to toluene.

The present study was initiated to examine whether urinary benzylmercapturic acid (or N-acetyl-S-benzyl cysteine, BMA), a mercapturate metabolite of toluene, increases in relation to the intensity of toluene exposure, and whether this metabolite is a better marker of occupational exposure to toluene than two traditional markers, hippuric acid and o-cresol. Accordingly, end-of-shift urine samples were collected from 122 printers and 30 office clerks (all men) in the second half of a working week. Solvent (toluene) exposure of the day (8 h) was monitored by means of diffusive sampling. Quantitative relation with toluene showed that BMA had a greater correlation coefficient with toluene (r = 0.7) than hippuric acid (r = 0.6) or o-cresol (r = 0.6). The levels in the urine of the non-exposed control subjects were below the detection limit of 0.2 microg/l for BMA, whereas it was at substantial levels for hippuric acid and o-cresol (239 mg/l and 32 microg/l as a geometric mean, respectively). Thus, BMA, hippuric acid and o-cresol could separate the exposed from the non-exposed when toluene was at < 1, 50 and 3 ppm, respectively. Overall, therefore, it appeared reasonable to conclude that BMA is superior to hippuric acid and o-cresol as a marker of occupational exposure to toluene.

Mechanism of the SDS-resistant synaptotagmin clustering mediated by the cysteine cluster at the interface between the transmembrane and spacer domains.

Synaptotagmin I (Syt I), a proposed major Ca(2+) sensor in the central nervous system, has been hypothesized as functioning in an oligomerized state during neurotransmitter release. We previously showed that Syts I, II, VII, and VIII form a stable SDS-resistant, beta-mercaptoethanol-insensitive, and Ca(2+)-independent oligomer surrounding the transmembrane domain (Fukuda, M., and Mikoshiba, K. (2000) J. Biol. Chem. 275, 28180-28185), but little is known about the molecular mechanism of the Ca(2+)-independent oligomerization by the synaptotagmin family. In this study, we analyzed the Ca(2+)-independent oligomerization properties of Syt I and found that it shows two distinct forms of self-oligomerization activity: stable SDS-resistant self-oligomerization activity and relatively unstable SDS-sensitive self-oligomerization activity. The former was found to be mediated by a post-translationally modified (i.e. fatty-acylated) cysteine (Cys) cluster (Cys-74, Cys-75, Cys-77, Cys-79, and Cys-82) at the interface between the transmembrane and spacer domains of Syt I. We also show that the number of Cys residues at the interface between the transmembrane and spacer domains determines the SDS- resistant oligomerizing capacity of each synaptotagmin isoform: Syt II, which contains seven Cys residues, showed the strongest SDS-resistant oligomerizing activity in the synaptotagmin family, whereas Syt XII, which has no Cys residues, did not form any SDS-resistant oligomers. The latter SDS-sensitive self-oligomerization of Syt I is mediated by the spacer domain, because deletion of the whole spacer domain, including the Cys cluster, abolished it, whereas a Syt I(CA) mutant carrying Cys to Ala substitutions still exhibited self-oligomerization. Based on these results, we propose that the oligomerization of the synaptotagmin family is regulated by two distinct mechanisms: the stable SDS-resistant oligomerization is mediated by the modified Cys cluster, whereas the relatively unstable (SDS-sensitive) oligomerization is mediated by the environment of the spacer domain.

The C2A domain of double C2 protein gamma contains a functional nuclear localization signal.

The C2 domain was originally defined as a homologous domain to the C2 regulatory region of Ca2+ -dependent protein kinase C and has been identified in more than 50 different signaling molecules. The original C2 domain of protein kinase Calpha functions as a Ca2+ binding module, and the Ca2+ binding to the C2 domain allows translocation of proteins to phospholipid membranes. By contrast, however, some C2 domains do not exhibit Ca2+ binding activity because of amino acid substitutions at Ca2+ -binding sites, and their physiological meanings remain largely unknown. In this study, we discovered an unexpected function of the Ca2+ -independent C2A domain of double C2 protein gamma (Doc2gamma) in nuclear localization. Deletion and mutation analyses revealed that the putative Ca2+ binding loop 3 of Doc2gamma contains six Arg residues ((177)RLRRRRR(183)) and that this basic cluster is both necessary and sufficient for nuclear localization of Doc2gamma. Because of the presence of the basic cluster, the C2A domain of Doc2gamma did not show Ca2+ -dependent phospholipid binding activity. Our findings indicate that by changing the nature of the putative Ca2+ binding loops the C2 domain has more diversified function in cellular signaling than a simple Ca2+ binding motif.

A simple HPLC method to determine urinary phenylmercapturic acid and its application to gasoline station attendants to biomonitor occupational exposure to benzene at less than 1 ppm.

The objective of this study was to establish a hand-saving method to measure phenylmercapturic acid (PMA) and to examine urinary PMA as a marker of occupational exposure to benzene at levels less than 1 ppm. A simple HPLC method was developed to analyse PMA by monitoring absorption at 195 nm of the ef? uent from an ODS-3 column with acetonitrile-methanol-perchloric acid-water as a mobile phase. The detection limit of the method was 0.2 µg l(-1) with sufficient reproducibility. The method was applied to end-of-shift urine samples from 70 gasoline station attendants exposed to up to 107 ppb benzene, and 20 non-exposed controls of both sexes. Time-weighted average (TWA) exposure to benzene was measured by diffusive sampling. A regression analysis was applied to examine the quantitative relationship between the intensity of exposure to benzene and PMA in the end-of-shift urine samples. Multiple regression analysis showed no effects of age, sex, smoking and co-exposure to toluene and xylenes on urinary PMA. There was a linear relationship between TWA benzene exposure and urinary PMA (r = 0.60-0.67, P < 0.01). Background PMA in urine of the non-exposed controls was low and scattering of PMA around the regression line was narrow so that those with 20 ppb benzene exposure can be separated from the non-exposed by urinalysis for PMA. Thus, urinary PMA is sensitive enough for biological exposure monitoring of those exposed to less than 1 ppm benzene.

Urinary phenylmercapturic acid as a marker of occupational exposure to benzene.

A hand-saving HPLC method to measure urinary phenylmercapturic acid (PMA) was developed which allows about 35 PMA determinations per day. The method involves conversion of pre-PMA to PMA by the addition of sulfuric acid to a urine sample, extraction into an ether-methanol mixture followed by condensation under a nitrogen stream. The condensate was introduced to a ODS-3 column in a HPLC system, and PMA in the column was eluted into a mobile phase of acetonitrile: methanol: perchloric acid: water. The elution of PMA was monitored at 205 nm. One determination will be completed in 40 min. The method was applied to analysis of end-of-shift urine samples from 152 workers exposed up to 210 ppm benzene, 66 workers exposed to a mixture of benzene (up to 116 ppm) and toluene + xylenes (up to 118 ppm), and 131 non-exposed controls of both sexes. A linear regression was established between time-weighted average intensity of exposure to benzene and urinary PMA. From the regression, it was calculated that urinary PMA level will be about 6.4 mg/l after 8-hour exposure to benzene at 100 ppm, and that PMA in urine accounted for about 0.1% of benzene absorbed. No effects of sex, age, and smoking habit of individuals were detected, and the effect of co-exposure to toluene + xylenes at the levels comparable to that of benzene was essentially nil, which indicates an advantage of PMA as a benzene exposure marker over monoto tri-phenolic metabolites or t,t-muconic acid.

Conserved N-terminal cysteine motif is essential for homo- and heterodimer formation of synaptotagmins III, V, VI, and X.

The synaptotagmins now constitute a large family of membrane proteins characterized by one transmembrane region and two C2 domains. Dimerization of synaptotagmin (Syt) I, a putative low affinity Ca(2+) sensor for neurotransmitter release, is thought to be important for expression of function during exocytosis of synaptic vesicles. However, little is known about the self-dimerization properties of other isoforms. In this study, we demonstrate that a subclass of synaptotagmins (III, V, VI, and X) (Ibata, K., Fukuda, M., and Mikoshiba, K. (1998) J. Biol. Chem. 273, 12267-12273) forms beta-mercaptoethanol-sensitive homodimers and identify three evolutionarily conserved cysteine residues at the N terminus (N-terminal cysteine motif, at amino acids 10, 21, and 33 of mouse Syt III) that are not conserved in other isoforms. Site-directed mutagenesis of these cysteine residues and co-immunoprecipitation experiments clearly indicate that the first cysteine residue is essential for the stable homodimer formation of Syt III, V, or VI, and heterodimer formation between Syts III, V, VI, and X. We also show that native Syt III from mouse brain forms a beta-mercaptoethanol-sensitive homodimer. Our results suggest that the cysteine-based heterodimerization between Syt III and Syt V, VI, or X, which have different biochemical properties, may modulate the proposed function of Syt III as a putative high affinity Ca(2+) sensor for neurotransmitter release.

High-pressure liquid chromatographic determination of toluene in urine as a marker of occupational exposure to toluene.

OBJECTIVE: To establish a convenient method by high-pressure liquid chromatography (HPLC) to measure toluene in urine as a marker of occupational exposure to toluene. METHODS: As soon after sampling as possible, 1 ml of urine was mixed with an equal volume of acetonitrile in a 2.2-ml HPLC glass bottle, and the bottle was tightly sealed and stored at 4 degrees C. Immediately before HPLC determination, 100 microl methanol was added to the mixture to prevent confounding effects of glycosuria, and the bottle was spun to remove any suspended matter. An aliquot of the supernate was introduced into the HPLC system and analyzed on a PRODIGY column, with an acetonitrile - perchloric acid phosphoric acid - water mixture serving as the mobile phase. The effluent was monitored at 191 nm. RESULTS: The method can measure toluene in urine every 20 min, the detection limit was 2 microg/l, the coefficient of variation was less than 5%, and the recovery rate was 100%. No significant reduction in toluene concentration was observed for 1 week after storage at 4 degrees C. When the method was applied to end-of-shift urine samples from 13 male workers exposed to toluene at 18-140 ppm and also to urine samples from 10 nonexposed male controls, toluene in urine was linearly related to toluene exposure concentration, with a regression line passing close to the origin. The correlation coefficient was as high as 0.97 (n=23). No toluene was detected in control urine samples. Calculations suggest that urinary toluene accounts for as little as less than 0.01% of the toluene absorbed via inhalation and that the absorbed toluene is converted almost quantitatively to hippuric acid and, by less than 0.1%, to o-cresol.

Retrospective study on the usefulness of radius and lumbar bone density in the separation of hemodialysis patients with fractures from those without fractures.

We measured bone mineral density (BMD) at the lumbar spine (LS-BMD), 1/3 radius (1/3R-BMD), and ultradistal radius (UDR-BMD) in 59 men (4 with spine fractures and 4 with nonspine fractures) and 65 women (10 with spine fractures and 9 with nonspine fractures), all receiving maintenance hemodialysis (HD). The BMD at each site expressed absolutely in g/cm2 was significantly lower in women than in men (p = 0.0001). In men, the absolute and age-matched values of both 1/3R- and UDR-BMD were inversely and significantly correlated with the duration of HD, and with serum alkaline phosphatase and parathyroid hormone levels (p < 0.05), whereas such relationships were obscure in women. On the other hand, the absolute values of BMD at each site in women but not in men were inversely and significantly correlated with patient age (p < 0.001). In both sexes, R-BMD was significantly lower in both the spine and nonspine fracture groups than in the nonfracture group (p < 0.05 and p < 0.01, respectively), whereas the only significant difference in LS-BMD was that it was lower in women with spine fractures than in women without fractures, when expressed as its absolute value (p < 0.05). By receiver operating characteristic analyses, both the absolute and age-matched values of R-BMD were better than LS-BMD as a determinant of non-spine fracture histories, and were similar to absolute LS-BMD as a determinant of spine fracture histories. We conclude that R-BMD is more valuable than LS-BMD for discriminating HD patients with all types of fractures from those without fractures.

[Lipoma in the lower lip: report of a case].

Lipoma, well-circumscribed tumors of mature adipose tissue, are one of the commonest encountered benign neoplasms. In the oral cavity, lipomas almost always occur in the cheek, tongue, and floor, but rarely in the lips. In this paper a 73-year old woman with lipoma in the right lower lip and cases of lipoma in the oral regions that have been reported in Japan are discussed. Her past history was diabetes mellitus and the others had no abnormalities. The specimen was a spherical, encapsulated, soft, bright-yellowish mass measuring 1.5 x 1.0cm. Microscopically, the tumor consisted of matured adipose tissue histological diagnosis of the tumor was simple lipoma. Six months postoperatively there is no sign of recurrence.