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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.

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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.

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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.

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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.

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Secretory leukocyte protease inhibitor (SLPI) and elafin are structurally similar, low-molecular-weight antiproteases produced in the lung. We have developed a simple method for distinguishing the antiprotease activities of SLPI and elafin in lung lavage fluid from those of alpha 1-antitrypsin (alpha 1-AT) that is based on the resistance of the low-molecular-weight antiproteases to inactivation by cetyltrimethylammonium bromide. In a study of 23 healthy, nonsmoking volunteers, we found that the low-molecular-weight antiproteases accounted for 22 +/- 2% (mean +/- SEM, n = 23) of the total neutrophil elastase-inhibitory capacity of human bronchoalveolar lavage fluid (BALF). Elafin activity was below the limit of detection. SLPI activity (as measured by inhibition of alpha-chymotrypsin) accounted for 72 +/- 4% (mean +/- SEM, n = 23) of the low-molecular-weight antiprotease activity in BALF. Measurements of SLPI in the lavage fluid samples by enzyme-linked immunosorbent assay (ELISA) agreed closely with values obtained by measuring the activity of this inhibitor. The activity of the low-molecular-weight antiproteases decreased significantly (p < 0.05), from 9.0 +/- 0.8 to 7.0 +/- 0.6 pmol of neutrophil elastase inhibited per mL (mean +/- SEM, n = 23), following acute ozone exposure.

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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.

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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.

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The aim of this study was to evaluate the possibility of applying low-intensity He-Ne laser irradiation as part of a complex system of anesthetic care of patients during invasive surgery. The following technique of intravenous low-intensity laser irradiation (i.v. LILI) was used. A filament of He-Ne laser was introduced through the subclavian venous line into the ostium of the vena cava superior. The power output was 20 mV, the exposure period 30 minutes. The irradiation began 10-15 minutes before anesthesia was introduced. In some cases, a second session was required. The 61 patients enrolled in the study were divided into a study group of patients who were irradiated and into a control group of patients who did not receive i.v. LILI. It was shown that i.v. LILI decreases neuroautonomous strain on the patient. Under the influence of i.v. LILI the P 50 appears to increase, the index of tissue oxygen extraction does not change, and the data of the acid base balance tend to improve. Low plasma levels of 11 oxycorticosteroids and the plasma content of 5-HTA and histamine as well as moderate changes in plasma enzyme activity confirm the efficacy of protecting patients from surgical trauma by application of i.v. LILI.

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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.

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Acute exposures to ozone have previously been shown to cause quantitative changes in the spectrum of arachidonic acid (AA) metabolites in lung lavage fluid. Since age appears to be an important variable in the toxicity of inhaled ozone, we investigated its effect on ozone-induced changes in pulmonary eicosanoid metabolism. Rats and rabbits ranging in age from neonates to young adults were exposed either to air or to 1 ppm ozone for 2 hr. Lung lavage fluid was collected within 1 hr following exposure and analyzed for its content of selected eicosanoids. In both species, there was a pronounced effect of age on ozone-induced pulmonary eicosanoid metabolism. Ozone-exposed animals at the youngest ages examined had severalfold greater amounts of two products of the cyclooxygenase pathway, prostaglandin E2 (PGE2) and prostaglandin F2 alpha (PGF2 alpha), than did age-matched controls. This effect lessened and eventually disappeared as the animals grew toward adulthood. In rabbits, ozone also induced increases in 6-keto-prostaglandin F1 alpha and thromboxane B2, but these changes were of lesser magnitude and evident only in the youngest rabbits exposed. There was no observed effect of ozone on lung lavage content of leukothriene B4. Indices of nonspecific pulmonary damage, i.e., protein concentration in lung lavage fluid and total number and viability of lavaged lung cells, were affected by ozone exposure, but not in an age-dependent manner that correlated with changes in pulmonary eicosanoid metabolism. In vitro ozone exposure of lung macrophages from naive rabbits of the same age range as those exposed in vivo demonstrated that ozone is capable of stimulating the elaboration of PGF2 alpha and especially PGE2. However, the increase in lavage fluid PGE2 and PGF2 alpha caused by ozone inhalation could not be attributed to macrophage metabolism conclusively since elaboration of PGE2 and PGF2 alpha by cultured macrophages was not enhanced by prior in vivo ozone exposure. In an ancillary study it was shown that 15-hydroxyprostaglandin dehydrogenase (PGDH) activity in rabbit lung homogenates was not affected by prior exposure to ozone, indicating that the increase in lung lavage fluid eicosanoids that occurred in these animals could not be explained by inhibition of PGDH.

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Inhaled acidic sulfate aerosols affect various aspects of lung function, presumably by delivery of hydrogen ion (H+) to target sites. Recent evidence suggests that the relationship between response and H+ content of the exposure atmosphere may depend upon the specific sulfate species with which the H+ is associated. This study examined comparatively the effects of exposure to the two main ambient acidic sulfates, sulfuric acid (H2SO4) and ammonium bisulfate (NH4HSO4), using the phagocytic activity of alveolar macrophages as the endpoint. Rabbits were exposed to 250-2000 micrograms/m3 H2SO4 (as SO4(-2)) and 500-4000 micrograms/m3 NH4HSO4 (as SO4(-2)) for 1 hr/day for 5 days; bronchopulmonary lavage was then performed for recovery of free lung cells. Phagocytosis, measured by uptake of opsonized latex spheres in vitro, was altered by exposure to H2SO4 at concentrations greater than or equal to 500 micrograms/m3 and to NH4HSO4 at greater than or equal to 2000 micrograms/m3. Assessment of results in terms of the calculated hydrogen ion concentration in the exposure atmosphere showed that identical levels of H+ produced different degrees of response depending upon whether exposure was to H2SO4 or NH4HSO4. On the other hand, macrophages incubated in acidic environments in vitro responded similarly regardless of whether H2SO4 or NH4HSO4 was used to adjust the pH. Possible reasons for the difference in response observed in vivo and in vitro are discussed. Speciation of ambient acidic sulfate aerosols may be needed in atmospheric monitoring so as to assess the presence of H+ posing the greatest biologic hazard following inhalation exposure.

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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.

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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.

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