Inserting Extrahelical Structures into Long DNA Substrates for Single-Molecule Studies of DNA Mismatch Repair.

The DNA mismatch repair (MMR) system corrects errors that occur during DNA replication. MMR needs the coordinated and highly dynamic assembly of repair enzymes at the site of the lesion. By visualizing transient intermediates of these assemblies, single-molecule approaches have shed critical insights into the mechanisms of MMR. These studies frequently require long (>20kb) DNA substrates with lesions and other extrahelical structures inserted at defined positions. DNA derived from bacteriophage λ (λ-DNA) is a high quality long (48.5kb) DNA substrate that is frequently used in single-molecule studies. Here we provide detailed protocols for site-specific incorporation of recombinant sequences and extrahelical structures into λ-DNA. We also describe how to assemble DNA curtains, and how to collect and analyze single-molecule observations of lesion recognition by MMR proteins diffusing on these DNA curtains. These protocols will facilitate future single-molecule studies of DNA transcription, replication, and repair.

[A Hebrew version of the FLACC scale: measurement of pain in non-verbal children].

BACKGROUND: The Israeli Ministry of Health requires that every patient have their pain routinely and systematically measured when there are treated in any of the country's medical institutions. Measurement guides treatment and enables follow-up of pain over time. Self-assessment of pain is the gold standard. Measurement is standardized by using scales representing intensity from "no pain" to "unbearable pain". Three-year-olds can assess their own pain, but younger children, or those who are non-verbal due to a medical procedure, cannot. Scales for this population rely on behavioral and physiological parameters, with assessment conducted by caretakers. Of the scales reviewed by the authors, the "FLACC" was chosen as appropriate for routine use due to its brevity and simplicity. AIMS: Translation and establishment of reliability and validity of the Hebrew version of the FLACC. METHODS: Subjects included 53 children aged 2 months to 8 years, who could not communicate verbally. Observations were conducted in the post-anesthesia care unit and the Intensive Care Unit. The FLACC was translated by the translation-back-translation technique. Inter-rater reliability was tested by two independent observers, and validity was assessed before and after provision of intravenous morphine or ketorolac. RESULTS: Inter-rater reliability was high for the total FLACC score (r = 0.94, p < 0.001), as well as for the separate items (kappa 0.5-0.85). The FLACC was considered valid, as the change in scores paralleled the known pharmacological effect of the medications. CONCLUSIONS: The Hebrew version of the FLACC was found reliable and valid for caretakers to use with children who cannot communicate verbally.

The geriatric mania asenapine study (GeMS).

RATIONALE: Population aging results in growing numbers of psychiatric disorders among older patients. Yet, there is a paucity of studies on elderly mania. OBJECTIVE: To evaluate the effect of asenapine on older manic inpatients. METHODS: Thirty-four elderly patients suffering from a manic episode, mean age 67.2 years were enrolled in an open-label 3-weeks study of asenapine treatment. INCLUSION CRITERIA: (1) DSM-IV criteria for manic episode (2) age above 60 years, (3) episode severity necessitating inpatient treatment, (4) Young Mania Rating Scale (YMRS) score at baseline >20, and (5) no prior asenapine treatment. Participants were prescribed asenapine 5 mg BID for 3 days and then dose increased to 10 mg BID till day 21 (study completion). RESULTS: Twenty-five patients completed the study. YMRS score decreased from a baseline mean of 27.0±8.8 to 13.3±12.0 at the end of the study (p<0.001). Fourteen patients (56% of completers) achieved remission (YMRS score<12). MADRS score decreased from a baseline mean of 7.6±5.6 to 4.4+5.1 at the end of the study (p<0.05); low baseline score should be noted. Sleep duration increased from a baseline median of 5.7 hours to 7.0 h at the end of the study (p<0.05). Seven patients discontinued treatment due to adverse events. Two patients passed-away after study completion. CONCLUSION: We tentatively conclude that the efficacy of asenapine in reducing acute manic symptoms and achieving remission in the elderly is supported in this study. Caution is needed in patients with co-morbid physical conditions.

Quantitative analysis of collagen content and amino acids in trabecular meshwork.

The purpose of this study was to compare collagen content in the TM of normal and glaucomatous eyes, and to establish whether collagen levels change with age. Collagen content was measured in 30 normal and 27 age matched glaucoma trabeculectomy specimens by the sirius red dye binding technique, and in 14 normal and 15 age matched glaucoma specimens by amino acid analysis. Both dye binding data and amino acid analysis showed no statistical difference between normal and glaucoma samples. Age had no significant effect on mean optical densities or on the collagen-specific amino acids proline, hydroxyproline, and hydroxylysine. Amino acid variability, however, was statistically different between the two groups. These results indicate that mean collagen levels in the trabecular meshwork of glaucomatous eyes do not differ from those in normal eyes.

Potential value of collagen shields as a subconjunctival depot release system.

Collagen shields are fabricated from dissoluable porcine scleral tissue and have been used as an ocular drug delivery system. The aim of the present study was to determine the time and extent of shield absorption when implanted subconjunctivally, and the absorption and release of 5-fluorouracil in vitro. Thirty New Zealand white rabbit eyes were employed. BioCor 72 hour collagen shields were surgically implanted in the subconjunctival space. Rabbits were sacrificed at 7, 14 and 21 days after shield implantation, and the remaining shields removed. Remaining shields were measured by both dry weight and protein assay. The absorption and release of 5-FU from collagen shields was determined in vitro using tritiated 5-FU. The collagen shields were not fully absorbed for at least 14 days in the subconjunctival space. In vitro, 5-FU absorbed by the shields reached saturation levels at approximately 15 minutes. Nearly 100% of the 5-FU was released within 15 minutes. Although the time for subconjunctival shield absorption may be useful for antifibroblast drugs, the rate of 5-FU release from these shields is not optimal for enhancing bleb formation when shields are soaked in solutions of 5-FU.

Lifetime needle sharing: a predictive analysis.

Through understanding predictors of needle sharing, it may be possible to design AIDS prevention interventions more effectively. Data were collected from a sample of 416 patients in two New York City methadone programs in 1990. Questions were asked about needle sharing and about a battery of predictors covering 11 psychosocial domains. Based on factor analysis, these were reduced to seven factors: criminal history, antisocial characteristics, social integration, severity of psychiatric problems, current drug involvement, physical health, and personality disorders. Bivariate analyses showed that criminal involvement, antisocial characteristics, social integration, and age were significantly related to needle sharing. With the seven factors, as well as age, gender, and ethnicity simultaneously examined by means of regression analysis, it was found that criminal involvement, severity of psychiatric problems, and age were all positively associated with needle sharing. Implications for treatment are discussed.

Further evaluation of collagen shields as a delivery system for 5-fluorouracil: histopathological observations.

Collagen shields are a potential delivery system for antifibroblast drugs such as 5-fluorouracil after filtration surgery. To determine whether collagen shields produce histologic evidence of inflammation when implanted subconjunctivally, shields were implanted into four rabbit eyes and six guinea pig eyes and retained for 7 or 14 days. Two rabbit eyes and two guinea pig eyes served as controls. Seven days after implantation in the rabbit eyes foreign-body giant cells were present at the surface of the shield, and early deposition of connective tissue was evident around the shield. The inflammatory response at 14 days was similar but more intense. In the guinea pig eyes the collagen shields induced substantially less inflammation, and there was marked shield degradation at 14 days. The results suggest that the inflammatory response in rabbits may be species specific and that collagen shields may be of value as a drug-delivery system for antifibroblast drugs in other species.

Inhibition of neutrophil elastase by mucus glycoprotein.

A number of structurally diverse polyanions have been found to inhibit human leukocyte elastase (HLE) activity. The purpose of this study was to examine the effects of mucus glycoprotein (mucin), one of the most plentiful high molecular weight polyanions in the respiratory tracts, on HLE activity. Human airway mucin and bovine submaxillary mucin at concentrations of 0.4 to 2.8 mg/ml both markedly inhibited the elastolytic activity of 50 nM HLE, with maximum inhibition approaching 90%. The degree of inhibition was the same regardless of whether the mucin, elastase, and elastin were simultaneously combined or whether the mucin was added to elastase 20 min prior to adding elastin, indicating that mucin is a rapid-acting inhibitor. The shape of the inhibition curve resembled that of curves obtained using heparin and HLE. Mucin had little inhibitory effect on pancreatic elastase, which is structurally related to but less cationic than HLE. The inhibition of HLE by mucin was blocked by 1 M NaCl. Removal of sulfate esters by acid-catalyzed solvolysis markedly reduced the inhibitory effect of bovine submaxillary mucin. These results indicate that inhibition of HLE by mucin involves binding of the positively charged HLE molecules to the negatively charged sulfated carbohydrates in the mucin. Mucin was also found to substantially reduce the antiprotease activity of secretory leukocyte protease inhibitor, a low molecular weight cationic protein known to bind to mucin.

Rat lung phospholipid fatty acid composition in prepregnant, pregnant, and lactating rats: relationship to ozone-induced pulmonary toxicity.

Our laboratory has demonstrated recently that pulmonary inflammation induced by acute ozone exposure is much more severe in late stage pregnant and lactating rats than in postlactating rats or age-matched virgin females. It is currently widely believed that such pulmonary damage results, at least in part, from the reaction of ozone at sites of unsaturation in phospholipid fatty acid (PLFA) molecules located in the epithelial fluid layer lining the lung surfaces and/or the plasma membranes of epithelial cells underlying this fluid layer. The objective of this study was to compare the PLFA composition of lung tissue and surfactant from ozone-sensitive late stage pregnant and lactating rats with comparable tissue from relatively ozone-insensitive age-matched prepregnant (virgin female) rats to explore the possibility that changes in lung PLFA composition during pregnancy and/or lactation contribute to the enhanced sensitivity of these physiologic states to ozone. In addition, the correlation of changes in plasma PLFA composition with those in lung was investigated. There were minor differences in the composition of lung tissue and surfactant PLFAs between prepregnant rats and pregnant rats at day 17 of gestation and only slightly greater differences between prepregnant and lactating rats. Changes from the prepregnant state in the PLFA composition of lung tissue, but not surfactant, correlated with changes in the plasma only in lactating rats and not in pregnant rats. Overall, the double bond index of PLFAs in surfactant and lung tissue was decreased in pregnant and lactating rats compared with prepregnant rats. Thus, the increased sensitivity of pregnant and lactating rats to ozone-induced lung injury cannot be attributed to an increased availability of unsaturated fatty acids. In addition, the arachidonic acid composition of phospholipids did not appear to explain differences between prepregnant rats and pregnant or lactating rats in their inflammatory response to ozone. In conclusion, there is no evidence that the relatively minor changes in lung tissue PLFA composition which occur during pregnancy and lactation predispose rats in these physiologic states to ozone-induced pulmonary toxicity.