Catalytic and thermodynamic properties of the urocanate hydratase reaction.

Urocanate hydratase (4-imidazolone-5-propionate hydro-lyase, EC 4.2.1.49) isolated from Pseudomonas putida contains covalently bound alpha-ketobutyrate as its cofactor. In the process of examining the mechanism by which alpha-ketobutyrate serves in this capacity, various thermodynamic parameters and temperature effects on urocanate hydratase activity were determined. As the equilibrium constant at 15 degrees C for imidazooone propionate formation from urocanate is approximately 69, regardless of whether urocanic acid or chemically synthesized imidazolone propionate is used as the initial substrate, it is concluded that the reaction is freely reversible. DeltaG degrees ', deltaH degrees ' and deltaS degrees ' were --2.5 kcal/mole, +5.2 kcal/mole and +26 cal/deg mole, respectively. Measurement of first-order reaction rates at various temperatures, in order to calculate the Arrhenius activation energy, showed a sharp break in the Arrhenius plot at 29 degrees C. Further examination of this phenomenon by determining s20,w values of urocanate hydratase as a function of temperature revealed a dramatic change at 31 degrees C. Since the enzyme in both experiments reverts to its original state when the temperature is lowered back below the transition point, it is proposed that urocanate hydratase undergoes a reversible conformational change or partial dissociation which affects its catalytic properties in the range of 29--31 degrees C.

Comparison of photosensitized plasma membrane damage caused by singlet oxygen and free radicals.

The efficiency and selectivity of photosensitized damage to membrane functions may be influenced strongly by the identity of the initial reactive species formed by the photosensitizer. To test this possibility, a photosensitizer, rose bengal (RB), was used that resides in the plasma membrane and which generates singlet molecular oxygen (1O2*) upon excitation with visible light, and radicals plus 1O2* upon excitation with UV radiation. With this approach, 1O2* and radicals are formed at the same locations in the plasma membrane. The response of three plasma membrane functions, namely, proline transport, membrane potential, and membrane impermeability to charged dye molecules, was assessed. The efficiencies of the responses in the presence and absence of oxygen were compared per photon absorbed by RB at two wavelengths, 355 nm (UV excitation) and 532 nm (visible excitation). The efficiency of oxygen removal before irradiation was assessed by measuring the RB triplet lifetime. The three membrane functions were inhibited more efficiently at 355 nm than at 532 nm in the presence of oxygen indicating that the radicals are more effective at initiating damage to membrane components than 1O2*. The ratio of photosensitized effects at the two wavelengths in the presence of oxygen was the same for two membrane functions but not for the third suggesting that 1O2* and radicals initiate a common mechanistic pathway for damage to some membrane functions but not to others. Removing oxygen reduced the efficiency of 355 nm-induced photosensitization by factors of 1.4 to 7. The sensitivity of the three membrane functions to 1O2*-initiated damage varied over a factor of 50 whereas radical initiated damage only varied by a factor of 15. In summary, these results indicate that radicals and 1O2* formed at the same locations in the plasma membrane vary in their efficiency and specificity for membrane damage but may, in some cases, operate by a common secondary damage mechanism in the presence of oxygen.

Benzoyl peroxide increases UVA-induced plasma membrane damage and lipid oxidation in murine leukemia L1210 cells.

Ultraviolet A radiation induces oxidative stress and cell damage. The purpose of this investigation was to examine whether ultraviolet A-induced cell injury was amplified by the presence of a non-ultraviolet A absorbing molecule capable of generating free radicals. Benzoyl peroxide was used as a lipid soluble potential radical-generating agent. Plasma membrane permeability assessed by trypan blue uptake was used to measure cell damage in murine leukemia L1210 cells. Cells were irradiated with a pulsed Nd/YAG laser at 355 nm using 0-160 J per cm2. The ratio of the fluence-response slope in the presence of 40 microM benzoyl peroxide to that of irradiated controls was 4.3 +/- 2.6. Benzoyl peroxide alone or benzoyl peroxide added after irradiation did not cause increased trypan blue uptake. The ratio of the fluence-response slopes in the presence of 40 microM benzoyl peroxide to that of irradiated controls was 4.7 +/- 1.4 when cells were irradiated (0-43 J per cm2) with a xenon lamp, filtered to remove wavelengths <320 nm. The increased trypan blue uptake in 355 nm-irradiated cells in the presence of benzoyl peroxide was inhibited in a concentration-dependent manner by butylated hydroxytoluene, vitamin E, and trolox, a water-soluble vitamin E derivative. Lipid oxidation, assessed as thiobarbituric acid reactive substances, was significantly increased in samples irradiated with ultraviolet A in the presence of benzoyl peroxide at fluences >34 J per cm2. The increased trypan blue uptake and thiobarbituric acid reactive substances were inhibited by butylated hydroxytoluene. These results suggest that agents not absorbing ultraviolet A radiation may enhance ultraviolet A-initiated oxidative stress in cells.

Activation of protein kinase C is required for protection of cells against apoptosis induced by singlet oxygen.

We evaluated the role of protein kinase C (PKC) in the regulation of apoptosis triggered by singlet oxygen. Activation of PKC by short-term 12-O-tetradecanoyl phorbol 13-acetate (TPA) treatment inhibited apoptosis, whereas inhibition of PKC with several inhibitors potentiated this process. The antiapoptotic effect of TPA was accompanied by phosphorylation of extracelluar signal-regulated kinase 1/2 (ERK1/2). Pretreatment of cells with MEK inhibitor, PD98059, inhibited TPA-induced phosphorylation of ERK1/2 and the cytoprotective ability of TPA. These results suggest that activation of PKC in HL-60 cells confers protection against apoptosis induced by singlet oxygen and that ERK1/2 mediates antiapoptotic signaling of PKC.

Singlet oxygen, but not oxidizing radicals, induces apoptosis in HL-60 cells.

Oxidizing species (OS), produced by photosensitization or derived from cytotoxic agents, activate apoptotic pathways. We investigated whether two different OS, formed at the same subcellular sites, have equivalent ability to initiate apoptosis in HL-60 cells. Our previous work showed that absorption of visible light by rose bengal (RB) produces singlet oxygen exclusively, whereas absorption of ultraviolet A produces RB-derived radicals in addition to singlet oxygen. Singlet oxygen, but not the RB-derived radicals, induced nuclear condensation and DNA fragmentation into nucleosome-size fragments in a dose dependent manner. In contrast, the RB-derived radicals caused greater lipid oxidation than singlet oxygen. These results indicate that different OS, produced at the same subcellular sites, do not have the same ability to induce apoptosis and that the ability of an OS to initiate lipid oxidation does not necessarily correlate with its ability to induce apoptosis.

An efficient oxygen independent two-photon photosensitization mechanism.

A novel oxygen-independent photosensitization mechanism from the upper triplet state (Tn) of rose bengal has been demonstrated by selectively populating Tn by sequential two-color laser excitation. Products formed from Tn inhibit red blood cell acetylcholinesterase and decrease viability of P388D1 mouse macrophage monocyte cells as measured by trypan blue exclusion assay. Laser flash photolysis studies indicate that Tn reacts efficiently, as evidenced by permanent photobleaching of T1 absorption, with chemical yields approaching unit efficiency. This mechanism may have application for oxygen deficient photosensitization under high intensity, pulsed laser irradiation.

Intensity-dependent enzyme photosensitization using 532 nm nanosecond laser pulses.

The intensity dependence of the rose bengal (RB)-photosensitized inhibition of red blood cell acetylcholinesterase has been studied experimentally and the results compared to a quantitative excitation/deactivation model of RB photochemistry. Red blood cell membrane suspensions containing 5 microM RB were irradiated with 532 nm, 8 ns laser pulses with energies between 1 and 98.5 mJ. A constant dose (7 J) was delivered to all samples by varying the total number of pulses. At incident energies greater than approximately 4.5 mJ/pulse, the efficiency for photosensitized enzyme inhibition decreased as the energy/pulse increased. The generation of RB triplet state was monitored as a function of laser energy and the triplet-triplet absorption coefficient was determined to be 1.9 x 10(4) M-1 cm-1 at 530 nm. The number of singlet oxygen molecules produced at each intensity was calculated from both the physico-mathematical model and from laser flash photolysis results. The results indicated that the photosensitized inhibition of acetylcholinesterase was exclusively mediated by singlet oxygen, even at the highest laser intensities employed.

Progression of active trachoma to scarring in a cohort of Tanzanian children.

Risk factors for the incidence of scarring are needed to inform trachoma control programs in countries hyperendemic for this blinding disease. A cohort of pre-school children with constant, severe trachoma, and an age, sex, and neighborhood matched cohort of children without constant severe trachoma were followed for seven years to determine the incidence of scarring. The incidence of scarring in the children with constant severe trachoma was 29.2% versus 9.6% in the comparison group. In a model adjusting for multiple factors, significant predictors of scarring were increasing age, female, and constant severe trachoma (OR = 4.85, 95% CL = 2.05, 11.40). Infection with C. trachomatis at follow up was also associated with scarring in both groups of children. It is likely that these children have a different host response to infection, and represent a subgroup at high risk for the blinding complications of trachoma. Reducing exposure to infection in the community through antibiotics and changes in hygiene practices is still the most promising control strategy.

Osteoid osteoma of a rib as a cause of scoliosis. A case report.

A case of osteoid osteoma of a rib causing painful scoliosis is reported. The importance of full investigation in such a case is highlighted.

Periosteal division and longitudinal growth in the tibia of the rat.

Growth at the proximal tibial epiphyseal plate of the rat has been measured following three different growth-stimulating procedures. These were proximal periosteal release, distal periosteal release and full periosteal stripping of the diaphysis. A new radiographic method using a photographic technique has made it possible to take accurate measurements of the rate of long-bone growth in small experimental animals. From the results of this animal series we conclude that proximal tibial periosteal division is likely to be the most effective of the three procedures when used to correct leg-length discrepancy in the growing child.

Facet joint injection for low back pain. A clinical study.

Therapeutic injection of facet joints is now widely practised, but British experience has been infrequently reported. We studied the results of injecting facet joints with a corticosteroid preparation in 50 patients suffering from the "facet syndrome". Our series included a number of extra-articular injections and these "failed injections" provide a useful control group. Results indicate that only intra-articular injections are effective; certainty of joint penetration can be ensured only by the routine use of joint arthrography.

Adjunctive benefit of an essential oil-containing mouthrinse in reducing plaque and gingivitis in patients who brush and floss regularly: a six-month study.

BACKGROUND: Mechanical methods of oral hygiene can be complemented by the use of chemotherapeutic mouthrinses. The authors sought to quantify the additional benefit provided by an essential oil-, or EO-, containing mouthrinse in reducing plaque and gingivitis in patients who brush and floss regularly. METHODS: The authors randomly assigned patients with gingivitis to one of three treatment groups: brushing and rinsing with a control mouthrinse, or BC; brushing, flossing and rinsing with a control mouthrinse, or BFC; or brushing, flossing and rinsing with an EO-containing mouthrinse, or BFEO. Patients received a dental prophylaxis at baseline, and the authors followed them for six months. RESULTS: Of 246 enrolled subjects enrolled in the study, 237 subjects were evaluable at the study's conclusion. After six months, the subjects using the BFEO regimen had statistically and clinically significant lower mean Modified Gingival Index, or MGI, scores and Plaque Index, or PI, scores than did subjects in the BC group (29.9 percent and 56.3 percent, respectively; P < .001). Subjects in the BFC group had statistically significantly lower mean MGI and PI scores than did subjects in the BC group (11.2 percent and 9.3 percent, respectively; P < .001). Subjects in the BFEO group exhibited statistically and clinically significantly lower mean scores for MGI and PI than did subjects in the BFC group (21 percent and 51.9 percent, respectively; P < .001). CONCLUSIONS: This study confirms that for patients with gingivitis who brush and floss routinely, the adjunctive use of an EO-containing mouthrinse provides a clinically significant and meaningful additional benefit in reducing plaque and gingivitis. CLINICAL IMPLICATIONS: An EO-containing mouthrinse is an effective adjunct to regular brushing and flossing. Therefore, the BFEO regimen can be beneficial for patients with gingival inflammation.

Percutaneous accessory collateral ligament release in the treatment of proximal interphalangeal joint flexion contracture.

A percutaneous release or capsulo-ligamentous proximal interphalangeal joint contracture is described, the indications are outlined and the advantage over open releases are discussed. The results of a small pilot study are reported which we feel are comparable to the results of open techniques. The procedure in itself, however, merely allows operative correction to be achieved. Its maintenance depends upon a stringent post-operative rehabilitation regime.

A new radiological method for assessing long bone growth in the small laboratory animal.

The growth of the immature rat tibia has been measured following five different growth stimulating procedures. These comprised proximal periosteal release, proximal periosteal release with insertion of dissimilar metal wire, repeated proximal periosteal release, distal periosteal release and full periosteal stripping of the diaphysis. The new radiographic method of measurement employed, utilizes a photographic technique. A reproducible measurement of the rate of long bone growth in the small experimental animal is possible.

Risks of occupational exposure to latex gloves.

The extraordinary increase in latex glove use in dentistry within the past decade has created a potential occupational hazard in the form of adverse reactions to components found in these gloves. Reactions may range from dry, itchy skin to a life-threatening, anaphylactic response. Management of these conditions may be as simple as switching glove brands; but in the most severe cases, it may entail the need to create a latex-free environment for the safety of the affected health care worker. This article reviews the pathophysiology, epidemiology, diagnosis and management of these conditions and provides references for more in-depth reading on the subject.

Tibia recurvatum as a complication of Osgood-Schlatter's disease: a report of two cases.

Premature fusion of the anterior part of the upper tibial epiphyseal plate is a rare complication of Osgood-Schlatter's disease. The resulting deformity is both unsightly and difficult to treat. We report two further cases and highlight the need for regular screening for this rare complication of a common disease.