Favorable clinical outcome and unique characteristics in association with Twist1 overexpression in de novo acute myeloid leukemia.

Epithelial-mesenchymal transition (EMT) is a critical process for inducing stem-like properties of epithelial cancer cells. However, the role of EMT inducers in hematological malignancies is unknown. Twist1, an EMT inducer necessary for cell migration, has recently been found to have transcriptionally regulatory activity on the expression of Bmi1, and these two are capable of promoting tumorigenesis in a synergized manner. Knowing that Bmi1 expression is essential for maintenance of leukemic stem cells, we speculate that Twist1 might govern the pathogenesis of acute myeloid leukemia (AML) development as well. We found that upregulated Twist1 increased Bmi1 expression in AML and endued leukemic cells a higher proliferative potential and increased resistance to apoptosis. In primary AML samples, there was strong positive correlation between the expression levels of Twist1 and Bmi1. AML patients whose leukemic blasts harbored overexpressed Twist1 had a more aggressive clinical phenotype, but they were more likely to have a better clinical outcome after standard therapy. In vitro studies confirmed that Twist1-overexpressing leukemic cells were more susceptible to cytarabine, but not daunorubicin, cytotoxicity. Our findings suggest that, in a subset of AML patients, Twist1 has a prominent role in the pathogenesis of the disease that leads to unique clinical phenotypes.

DAPK activates MARK1/2 to regulate microtubule assembly, neuronal differentiation, and tau toxicity.

Death-associated protein kinase (DAPK) is a key player in several modes of neuronal death/injury and has been implicated in the late-onset Alzheimer's disease (AD). DAPK promotes cell death partly through its effect on regulating actin cytoskeletons. In this study, we report that DAPK inhibits microtubule (MT) assembly by activating MARK/PAR-1 family kinases MARK1/2, which destabilize MT by phosphorylating tau and related MAP2/4. DAPK death domain, but not catalytic activity, is responsible for this activation by binding to MARK1/2 spacer region, thereby disrupting an intramolecular interaction that inhibits MARK1/2. Accordingly, DAPK(-/-) mice brain displays a reduction of tau phosphorylation and DAPK enhances the effect of MARK2 on regulating polarized neurite outgrowth. Using a well-characterized Drosophila model of tauopathy, we show that DAPK exerts an effect in part through MARK Drosophila ortholog PAR-1 to induce rough eye and loss of photoreceptor neurons. Furthermore, DAPK enhances tau toxicity through a PAR-1 phosphorylation-dependent mechanism. Together, our study reveals a novel mechanism of MARK activation, uncovers DAPK functions in modulating MT assembly and neuronal differentiation, and provides a molecular link of DAPK to tau phosphorylation, an event associated with AD pathology.

An interesting case of recurrent temporomandibular joint dislocation.

Acute temporomandibular joint dislocation is an infrequent presentation to the emergency department or to dental practitioners. It may be a rare complication of dental treatment. Recurrent temporomandibular joint dislocation occurs in individuals with certain anatomical abnormalities or systemic conditions. This paper reports an unusual presentation of recurrent temporomandibular joint dislocation and outlines some of the surgical techniques used in managing this condition.

Facial fractures in road cyclists.

BACKGROUND: Despite the safety devices worn by bicycle riders and the road safety measures designed to protect them, road cycling is still associated with frequent traumas, notably to the extremities. Maxillofacial injuries are common, especially when road traffic accidents occur. There is a paucity of literature on the epidemiology of bicycle-related facial fractures in New Zealand. METHODS: A retrospective database of patients presenting to the Oral and Maxillofacial Surgery Unit at Christchurch Hospital during an 11-year period was reviewed. Variables examined included demographics, types of fracture, mode of injury, and treatment delivered. RESULTS: Sixty-three patients were identified as having road bicycle-related facial fractures, with no increase in prevalence over the study period. Male to female ratio was 3:1, with 76 per cent involving patients aged between their first and third decade. Fall was the most common mechanism of injury, followed by collision. Fifty-two per cent of patients were hospitalized and 40 per cent required surgical intervention. The highest incidence of injury was during January. CONCLUSIONS: Road bicycle-related fractures accounted for a small proportion of the workload at our unit. They predominantly affected young male adults. More than half of the patients were hospitalized and a major proportion of these required surgery.

Determination of diethanolamine and N-nitrosodiethanolamine in fatty acid diethanolamides.

Diethanolamine (DEA) is a precursor of N-nitrosodiethanolamine (NDELA), an animal carcinogen. A gas chromatographic (GC) method was developed for determining DEA in fatty acid diethanolamides that are commonly used in cosmetic products. Methanolic solutions of the amides were analyzed by GC with flame ionization detection on either a wide-bore methyl silicone (Rtx-1) or 95% dimethyl--5% diphenyl polysiloxane (SPB-5) capillary column. Recovery of DEA from fatty acid dialkanolamides at fortification levels of 0.50, 1.00, and 5.00% ranged from 94 to 100%. In a survey of commercial fatty acid diethanolamides, DEA was found at levels ranging from 1.1 to 14.0%, and most were in good agreement with manufacturer's DEA specifications. Fatty acid diethanolamides also were anlayzed for NDELA by liquid chromatography interfaced to a thermal energy analyzer. Recovery of NDELA from fatty acid diethanolamides at fortification levels of 50, 100, and 200 ppb averaged 95%. No NDELA was found in any of the fatty acid diethanolamide samples analyzed.

A rapid and selective method for determining potential nitrosating agents in cosmetic products by chemiluminescence detection of nitric oxide.

A method was developed for rapid and selective determination of potential nitrosating agents at the part-per-billion level in cosmetic products. These compounds are chemically reduced to nitric oxide, which is determined by its chemiluminescent reaction with ozone. Suspended materials and colors in cosmetic products do not interfere. Hence their removal before analysis is not required. A detection limit of 33 ppb, calculated as nitrite, was obtained. No false-positive interferences were observed from antifoaming agents, several N-nitroso compounds, and nitrate up to 20 ppm. Among cosmetic products surveyed, potential nitrosating agents were found at levels ranging from 113 to 5021 ppb. No consistent relationship was found between levels of potential nitrosating agents and N-nitrosamines in the same products. However, the highest levels of nitrosating agents were most often associated with the highest levels of N-nitrosamines known to be present in the products.

Generation of complement membrane attack complex in normal human corneas.

PURPOSE: To determine whether complement-derived SC5b-9, the soluble nonlytic-fluid phase of the membrane attack complex, can be generated in normal human corneas when they are injured with lipopolysaccharide (LPS), ribitol teichoic acid (RTA) immune complexes, acid, or alkali. METHODS: The experimental cornea of each donor pair was injected with 50 microliters of sterile saline containing 0.5 mg of LPS or 50 microliters of sterile saline containing 250 micrograms of RTA immune complexes. Other experimental corneas were treated topically for 35 seconds with either 200 microliters of 1N HCl or 2N NaOH. The control cornea of each donor pair was injected with 50 microliters of sterile saline or was treated topically for 35 seconds with 200 microliters of sterile saline. After injury, all corneas were incubated in medium 199 for 6 hours at 37 degrees C in 5% CO2, then eluted for 24 hours in phosphate-buffered saline with 10 mM ethylenediaminetetraacetic acid. Each corneal eluate was collected and stored at -70 degrees C until assayed for SC5b-9 by an enzyme immunoassay. RESULTS: Compared with control corneas, SC5b-9 levels were increased significantly in corneas injected with LPS or RTA immune complexes. However, when compared with controls, SC5b-9 levels were decreased significantly in corneas treated with HCl or NaOH. CONCLUSIONS: Normal human corneas injured immunologically with LPS or RTA immune complexes activate the classical or alternate pathway and generate SC5b-9. Corneas injured chemically with acid or alkali do not produce SC5b-9.

Determination of 2-ethylhexyl 4-(N-methyl-N-nitrosamino) benzoate in commercial sunscreens and cosmetic products.

An analytical method has been developed for determination of 2-ethylhexyl 4-(N-methyl-N-nitrosamino) benzoate (NMPABAO), a nitrosamine contaminant in sunscreen products containing 2-ethylhexyl 4-(N,N-dimethylamino) benzoate (Padimate O). The method involves extraction of NMPABAO by column chromatography followed by liquid chromatographic separation and analysis wit a nitric oxide detector. To confirm the presence of NMPABAO in sunscreen products, the N-nitrosamine was synthesized and its structure was determined by infrared spectrophotometry, nuclear magnetic resonance spectrometry, and mass spectrometry (MS). For method validation, recovery studies were performed on a commercial suntan lotion, cream, and gel. Recoveries of NMPABAO added to representative test samples averaged 83%. The method has an estimated detection limit of 30 ppb. The method was used to analyze 25 commercial cosmetic and sunscreen products containing Padimate O. Eleven products contained NMPABAO at levels ranging from 160 to 21000 ppb. NMPABAO presence in 4 products was confirmed by MS at levels > or = 4000 ppb. The highest levels of NMPABAO were associated with products that contained the nitrite-releasing preservative 2-bromo-2-nitro-1,3-propanediol.

Complement system and host defense against staphylococcal endophthalmitis.

PURPOSE: The authors studied the role of the complement system in host defense against Staphylococcus epidermidis and S. aureus endophthalmitis. METHODS: Guinea pigs in the S. epidermidis model received an intravitreal injection of 7000 viable organisms, and guinea pigs in the S. aureus model received 50 viable organisms. The experimental animals in each model were decomplemented with intraperitoneal (IP) injections of cobra venom factor, whereas the control animals received IP injections of normal saline. Mean log bacterial counts in the vitreous and mean serum complement titers were compared in the experimental and control animals in each model on days 1, 2, 3, and 7. RESULTS: In the S. epidermidis model, mean log bacterial counts in the vitreous were significantly higher in the experimental group than the control group on days 1 and 2 (P < 0.01) and on day 3 (P < 0.05). Mean serum complement titers were significantly lower in the experimental group at all days (P < 0.01). In the S. aureus model, mean log bacterial counts in the vitreous were significantly higher in the experimental group than the control group on day 2 (P < 0.05) and day 3 (P < 0.01). Mean serum complement titers were significantly lower in the experimental group on days 1, 2, and 3 (P < 0.01), but not on day 7. CONCLUSION: These results suggest that decomplemented guinea pigs show impaired host defense to S. epidermidis and S. aureus endophthalmitis and that this defense is restored as complement levels approach normal.

Collagen shields impregnated with gentamicin-dexamethasone as a potential drug delivery device.

We investigated the ability of collagen shields impregnated with gentamicin sulfate and dexamethasone to deliver medication into rabbit eyes. We compared the aqueous humor gentamicin and dexamethasone levels delivered by collagen shield and subconjunctival injection therapy at five time points over a ten-hour period, by using a fluorescence polarization immunoassay and high-pressure liquid chromatography. Our in vitro studies showed that impregnated collagen shields released most of the gentamicin within the first 30 minutes of elution, whereas dexamethasone was released gradually over a ten-hour period. The collagen shields impregnated with gentamicin and dexamethasone produced aqueous gentamicin levels that were significantly lower (P = .014) than those produced by subconjunctival injection therapy at 30 minutes and that were comparable to subconjunctival injection therapy at the other time points. Minimum inhibitory concentrations of gentamicin (approximately 4 micrograms) were observed in aqueous humor within 30 minutes in rabbits that received subconjunctival injections and at one hour in rabbits treated with impregnated collagen shields. The impregnated collagen shields produced aqueous dexamethasone levels that were significantly lower (P = .004) than those produced by subconjunctival injection therapy at one hour, significantly higher (P = .028) than subconjunctival injection therapy at six hours, and comparable to subconjunctival injection therapy at the other time points. Collagen shield delivery of gentamicin-dexamethasone may be comparable to subconjunctival injections and provide an alternative therapy after intraocular surgery.

Prolongation of corneal allograft survival with liposome-encapsulated cyclosporine in the rat eye.

PURPOSE: To study the effects of different formulations of topical cyclosporine (Cyclosporin A [CsA]) on corneal allograft rejection in a rat model. METHODS: Female Lewis rats received penetrating keratoplasties from female Wistar-Furth donors. A total of 78 allogeneic grafts were performed. An additional 15 syngeneic grafts (Lewis) were used as technical controls. Two CsA preparations with equivalent drug concentrations (2.1 mg/ml) were applied as drops: CsA encapsulated in large unilamellar liposomes (CsA-LIP) and CsA dissolved in olive oil (CsA-DR). Allogeneic grafts were randomly assigned to receive CsA-LIP or CsA-DR beginning on the day of surgery five times daily for 10 days. Animals without any treatment or receiving empty liposomes (EM-LIP) were used as treatment controls. Grafts were graded three times weekly and a rejection index was generated based on graft clarity, neovascularization, and vessel size. RESULTS: All syngeneic grafts remained clear over the observation period of 60 days. Rejected allogeneic grafts without any treatment and those receiving EM-LIP or CsA-DR showed a mean survival time (+/- standard deviation) of 14 +/- 4, 14 +/- 5, and 14 +/- 4 days, respectively. There was no significant difference in mean survival time between the grafts without any treatment and those in CsA-DR or EM-LIP treatment groups. The mean survival time of rejected grafts in animals receiving CsA-LIP was prolonged to 20 +/- 4 days. There was a significant difference in the mean survival time between the CsA-LIP treatment group and groups receiving CsA-DR, EM-LIP, or no treatment (P < or = 0.05). The Kaplan-Meier survival curve of the CsA-LIP treatment group was significantly different from the other experimental groups. The graft survival rate in the CsA-LIP group was 77%, whereas the rate was 37% in the non-treated group, 45% in the CsA-DR group, and 36% in the EM-LIP group. CONCLUSION: Encapsulation of CsA in liposomes might be a promising formulation for use in the prevention of corneal graft rejection.

Screening cosmetic products for N-nitroso compounds by chemiluminescent determination of nitric oxide.

Cosmetic products were screened for total N-nitroso compounds by chemiluminescent measurement of nitric oxide liberated by the reductive cleavage of the N-nitroso group. The cosmetic was first partitioned between methylene chloride and water to separate polar and nonpolar N-nitroso compounds. Each extract was then examined for the presence of N-nitroso compounds by adding the cleavage reagent and sweeping the nitric oxide formed into a chemiluminescent analyzer. Although the method is not intended to be quantitative, recovery studies were conducted to determine measurable levels. Recovery studies of polar N-nitroso compounds were conducted by adding N-nitrosodiethanolamine (NDELA) to a cream, a shampoo, and a lotion at 3 levels, i.e., 80, 320, and 960 ppb, and then determining NDELA by the method. Recoveries ranged from 48 to 83% (mean 68%; SD = 11.9). For recoveries of nonpolar N-nitroso compounds, 100, 200, and 500 ppb of N-nitrosomethyltetradecylamine were added to the 3 cosmetic products. Recoveries ranged from 58 to 70% (mean 63%; SD = 5.3).

Pepstatin inhibition of proteinase-free human renin.

The kinetics of inhibition of proteinase-free human renin and of pepsin by the carboxyl proteinase inhibitor pepstatin have been examined. Inhibition was of the tight-binding type with both enzymes. Inhibition of crude human renin was of the classical, freely reversible type, but most of the renin-like activity of the preparation was due to contaminating proteinases which could be separated chromatographically from the renin. These results clarify previous kinetic studies of pepstatin inhibition of human renin.

Proteinase activity of human renin preparations: the International Reference Preparation (Renin Standard 68/356).

1. Several commonly used preparations of human renin, including the International Reference Preparation (Renin Standard 68/356), were examined for the presence of contaminating proteinase activity by using a 14C-glycinated bovine haemoglobin substrate assay. 2. All of the human renin preparations tested cleaved haemoglobin even in the presence o di-isopropylfluorophosphate and ethylenediaminetetra-acetate (EDTA). For a given amount of renin activity, varying amounts of proteinase activity were seen. The pH optimum also varied between preparations. 3. Small peptide inhibitors of human renin were not able to inhibit the proteinase activity. Furthermore a diazoacyl reagent and pepstatin, both potent inhibitors of aspartic acid-active site proteinases, were only partially inhibitory. 4. These and other observations suggest that the proteinase activity of the human renin preparations is not due to renin itself, but to contaminating proteinases of different types. Since these enzymes may produce angiotensin I or peptides which may interfere in renin assays, crude preparations of renin which contain proteinase activity, including the International Reference Preparation, should be used with caution or replaced by proteinase-free human renin which can be easily prepared by use of suitable affinity chromatography.

Separation of human renal renin and pseudorenin by affinity chromatography on hemoglobin-Sepharose-2B.

Human renal renin (EC 3.4.99.19) and pseudorenin were easily separated in a single step by affinity chromatography on hemoglobin-Sepharose-2B. Renin and pseudorenin were monitored by their actions on crude and partially purified hog protein renin substrates at neutral and acidic pH and on synthetic labelled polymeric renin substrate. Under the conditions employed (0.1 M sodium acetate (pH 3.5)/1 M sodium chloride at 4 degrees C) renin does not bind to the affinity adsorbent while pseudorenin is effectively bound and can be eluted only after raising the pH to 6.5. Pseudorenin-free renin prepared by this method is devoid of proteolytic activity toward hemoglobin. The chromatographic behaviour of renal pseudorenin on hemoglobin-Sepharose-2B is similar to that of cathepsin D.

Template specific inhibitors of E. coli RNA polymerase.

Electroneutral analogs of polynucleotides, poly-9-vinyladenine and poly-1-vinyluracil inhibit E. coli RNA polymerase in all combinations where the single stranded polynucleotides are used as templates and the vinyl analogs are complementary to them. As templates both the ribo- and deoxyribopolynucleotides were tested; variation of the template concentration in the presence of vinyl analogs produced a competitive pattern of inhibition. The electroneutral analogs do not inhibit the enzyme activity when non-complementary single stranded polynucleotides or double stranded polynucleotides are used as templates; the latter fully supports transcription even when one of the strands is complementary to the analog.