Structural basis for heterogeneous kinetics: reengineering the hairpin ribozyme

The RNA cleavage reaction catalyzed by the hairpin ribozyme shows biphasic kinetics, and chase experiments show that the slow phase of the reaction results from reversible substrate binding to an inactive conformational isomer. To investigate the structural basis for the heterogeneous kinetics, we have developed an enzymatic RNA modification method that selectively traps substrate bound to the inactive conformer and allows the two forms of the ribozyme-substrate complex to be separated and analyzed by using both physical and kinetic strategies. The inactive form of the complex was trapped by the addition of T4 RNA ligase to a cleavage reaction, resulting in covalent linkage of the 5' end of the substrate to the 3' end of the ribozyme and in selective and quantitative ablation of the slow kinetic phase of the reaction. This result indicates that the inactive form of the ribozyme-substrate complex can adopt a conformation in which helices 2 and 3 are coaxially stacked, whereas the active form does not have access to this conformation, because of a sharp bend at the helical junction that presumably is stabilized by inter-domain tertiary contacts required for catalytic activity. These results were used to improve the activity of the hairpin ribozyme by designing new interfaces between the two domains, one containing a non-nucleotidic orthobenzene linkage and the other replacing the two-way junction with a three-way junction. Each of these modified ribozymes preferentially adopts the active conformation and displays improved catalytic efficiency.     

Morphological transformation of C3H/M2 mouse fibroblasts by extracts of human mammary lipid

The aim of this study was to determine whether suckling-induced prolactin (PRL) levels were modified when milk ejection was impaired. Milk ejection impairment was achieved in two experimental models: a) depriving the dam of sleep during suckling and b) increasing the nonsuckling intervals in lactating dams. Sleep deprivation blocked milk ejection and enhanced suckling-induced PRL levels in dams that had been previously separated from their pups. When milk ejection is blocked in litter-deprived dams, mammary glands are not evacuated and they remain engorged. Suckling stimuli were not the cause of the difference in suckling-induced serum PRL levels in control and sleep-deprived dams. The engorgement of the mammary glands may play a role, as a maximum suckling-induced PRL increase was not observed in nonseparated SD dams with nonengorged mammary glands. Moreover, suckling-induced PRL levels were decreased when engorged mammary glands of SD dams were evacuated through an oxytocin injection. A parallel increase between suckling-induced PRL levels and mammary gland weight was observed in the experiments in which milk ejection was impaired through an increase in the intervals of nonsuckling, providing additional support for a relationship between mammary gland engorgement and the regulation of suckling-induced PRL levels.     

Lesion volume measurement in multiple sclerosis: how important is accurate repositioning?

This study was designed to estimate the contribution of repositioning inaccuracies to measured intracranial lesion volumes in multiple sclerosis (MS). Five patients with MS were each scanned 10 times, using spin-echo imaging (2,000/34/90), contiguous 5-mm slices, and different scanning positions. The maximum displacements from baseline were +/-4 degrees (AP rotation) and 3 mm (slice offset). Lesion volume was measured twice for each scan using a semiautomated contour technique. Measured lesion volumes ranged from 4,328 mm3 to 164,831 mm3. The mean intrarater coefficient of variation (CV) calculated for individual patients ranged from 1.1 to 4% (median, 1.7%). Using analysis of variance, the overall variance and CV due to altered scan position were greater than that due to rater error (repositioning CV 4.0%, intrarater CV 3.5%). The worst-case difference between volumes in the same patient ranged from 8.9 to 32% (median, 9.9%). Both rater and repositioning errors were greater for smaller lesion volumes. The maximum potential error due to repositioning inaccuracies is of a similar magnitude to the 5 to 10% expected change in lesion volume over 1 year. This study justifies continued careful attention to accuracy in repositioning for serial MR studies in patients with MS.     

The effect of recombinant human erythropoietin on hemostasis, fibrinolysis, and blood rheology in autologous blood donors

We report three cases of Castleman's disease mimicking the features of collagen disease. Case 1: A 39-year-old woman presented with intermittent arthralgia and fever. Laboratory findings were positive results for antinuclear antibody (80x speckled type), the LE test, anti-SSA antibody, anti-RNP antibody, and Coombs test. The patient was suspected to have systemic lupus erythematosus (SLE) or Sj?gren syndrome, but a lymph node biopsy revealed the plasma cell type of Castleman's disease. Steroid treatment led to resolution of her symptoms. Case 2: A 60-year-old man with mixed type Castleman's disease had proteinuria with renal dysfunction, autoimmune thrombocytopenia, antinuclear antibody, anti-RNP antibody, anti-DNA antibody and anti-cardiolipin antibody. The patient was suspected to have SLE but cervical lymph node biopsy revealed the mixed type of Castleman's disease. Symptoms were not controlled with steroid therapy. He developed renal failure that required for hemodialysis and died of gastrointestinal bleeding due to severe thrombocytopenia. Case 3: A 46-year-old woman had Raynaud's phenomenon, sclerodactylia, and nail fold bleeding. Laboratory tests were revealed positive for antinuclear antibody, anti-ENA antibody, and LE cell preparation. Radiographic study showed multiple masses in the retroperitoneal spaces, which necessitated laparotomy. Firstly, the patient was suspected to have systemic sclerosis or mixed connective tissue disease (MCTD). A biopsy revealed the hyaline-vascular type of Castleman's disease. The serum level of IL-6 by ELISA was high in all of three cases. In case 1, symptoms improved and the IL-6 level normalized after steroid treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spontaneous coronary artery dissection associated with oral contraceptive use

A 39-year-old woman presented with acute myocardial infarction due to spontaneous dissection of the left anterior descending artery. This was treated conservatively. She had been on combined oral contraceptive pills from the age of 22. Repeat coronary angiography 7 months later revealed spontaneous complete healing.     

Fluorescence properties of autofluorescent granules generated by cultured human RPE cells

PURPOSE: To compare the fluorescence properties of autofluorescent granules generated by retinal pigment epithelial (RPE) cells in vitro with those of the lipofuscin of RPE in vivo. METHODS: Cultured human RPE cells were maintained in basal medium for as long as 1 year, fed rod outer segments (ROS) daily for as long as 56 days, fed ROS in the presence and absence of leupeptin, or fed liposomes consisting of the major phospholipids in ROS. At different time points, cells were examined for overall fluorescence, and their fluorescence spectra were determined. In addition, chloroform-methanol extracts were examined by thin-layer chromatography and compared with those generated from RPE lipofuscin. RESULTS: Autofluorescent granules accumulated in cultured RPE cells, regardless of the presence of an exogenous substrate or the nature of the substrate. The rate of accumulation of autofluorescent granules was greatest in cells fed ROS. The autofluorescent material generated in cultured RPE cells had some spectral similarities with RPE lipofuscin but differed in solubility and chromatographic mobility of their constituent fluorophores. CONCLUSIONS. The autofluorescent granules generated by cultured RPE, even with different specific substrates, differ from lipofuscin granules in vivo, suggesting that additional properties of RPE cells or of the materials they phagocytose are required to produce autofluorescent materials with the characteristics of lipofuscin.     

Urokinase and the intestinal mucosa: evidence for a role in epithelial cell turnover

The enzyme nitric oxide synthase catalyzes the oxidation of the amino acid L-arginine to L-citrulline and nitric oxide in an NADPH-dependent reaction. Nitric oxide plays a critical role in signal transduction pathways in the cardiovascular and nervous systems and is a key component of the cytostatic/cytotoxic function of the immune system. Characterization of nitric oxide synthase substrates and cofactors has outlined the broad details of the overall reaction and suggested possibilities for chemical steps in the reaction; however, the molecular details of the reaction mechanism are still poorly understood. Recent evidence suggests a role for the reduced bound pterin in the first step of the reaction--the hydroxylation of L-arginine.     

Inattention as a key predictor of tobacco use in adolescence.

The authors examined the prediction from inattention to tobacco use among 2 cohorts (ages 7 and 13) of a community sample followed to young adulthood. Changes in self-reported tobacco use were tested with marginal transitional regression models, using parent and teacher ratings of inattention, hyperactivity-impulsivity, and other psychopathology, along with other factors, as predictors. Inattention, but not hyperactivity-impulsivity, significantly predicted adolescent tobacco use and young adult daily tobacco use. Peer substance use, parental substance use, and conduct disorder also predicted increases in tobacco use. African American ethnicity was strongly protective against later tobacco use. (PsycINFO Database Record (c) 2010 APA, all rights reserved)