Characterization of two distinct monoclonal antibodies specific for glial cell line-derived neurotrophic factor

The kinesin-related motor protein CHO1/MKLP1 was initially thought to be expressed only in mitotic cells, where it presumably transports oppositely oriented microtubules relative to one another in the spindle mid-zone. We have recently shown that CHO1/MKLP1 is also expressed in cultured neuronal cells, where it is enriched in developing dendrites [Sharp et al. (1997a) J. Cell Biol., 138, 833-843]. The putative function of CHO1/MKLP1 in these postmitotic cells is to intercalate minus-end-distal microtubules among oppositely oriented microtubules within developing dendrites, thereby establishing their non-uniform microtubule polarity pattern. Here we used in situ hybridization to determine whether CHO1/MKLP1 is expressed in a variety of rodent neurons both in vivo and in vitro. These analyses revealed that CHO1/MKLP1 is expressed within various neuronal populations of the brain including those in the cerebral cortex, hippocampus, olfactory bulb and cerebellum. The messenger ribonucleic acid (mRNA) levels are high within these neurons well after the completion of their terminal mitotic division and throughout the development of their dendrites. After this, the levels decrease and are relatively low within the adult brain. Parallel analyses on developing hippocampal neurons in culture indicate that the levels of expression increase dramatically just prior to dendritic development, and then decrease somewhat after the dendrites have differentiated. Dorsal root ganglion neurons, which generate axons but not dendrites, express significantly lower levels of mRNA for CHO1/MKLP1 than hippocampal or sympathetic neurons. These results are consistent with the proposed role of CHO1/MKLP1 in establishing the dendritic microtubule array.     

Induction and activation of mitogen-activated protein kinases of human lymphocytes as one of the signaling pathways of the immunomodulatory effects of morphine sulfate

Morphine sulfate causes immunomodulatory and immunosuppressive effects in human. In this study, the signaling pathway involved in these morphine effects was studied. Addition of morphine sulfate to human CEMx174 lymphocytic cells resulted in increased expression of mitogen-activated protein kinase cascade proteins. Morphine enhanced the cellular levels of ERK1 (44 kDa), ERK2 (42 kDa), a 54-kDa ERK, MEK1 (45 kDa), and MEKK (78 kDa). A time-dependent increase in the activated (Thr and Tyr dually phosphorylated) state of ERK1 and ERK2 was also observed. Naloxone, a morphine antagonist, reversed the observed morphine effects, implicating a micro opioid receptor-mediated process. These findings suggest that mitogen-activated protein kinases are important intermediates in signal transduction pathways initiated by morphine receptors in immune cells.     

Compartment-selective sensitivity of cardiovascular morphogenesis to combinations of retinoic acid receptor gene mutations

Several aspects of normal cardiovascular development require signaling by the vitamin A metabolite retinoic acid. We have previously established germ-line mutations in mice in the genes that encode the RAR alpha 1, RAR beta, and RXR alpha retinoic acid receptors as a means of studying the function of these receptors in vivo. Although mutation of RXR alpha results in fetal ventricular defects, the RAR alpha 1 and RAR beta mutations are apparently nonphenotypic in the heart and elsewhere. In this study, we have established and analyzed combinations of these receptor gene mutations. Malformations of the ventricular chamber (chamber hypoplasia and muscular ventricular septal defects), conotruncus (double-outlet right ventricle, transposition, and membranous ventricular septal defects), aortic sac (persistent truncus arteriosus and aorticopulmonary window), and aortic arch-derived arteries were recovered in various combinations of the RAR alpha 1, RAR beta, and RXR alpha gene mutations. Depending on the combination of receptor mutations, selective defects were obtained in specific cardiovascular compartments, suggestive of differential expression or function of each receptor within domains of the developing heart.     

Crystal structure and photodynamic behavior of the blue emission variant Y66H/Y145F of green fluorescent protein

The crystal structure of a blue emission variant (Y66H/Y145F) of the Aequorea victoria green fluorescent protein has been determined by molecular replacement and the model refined. The crystallographic R-factor is 18.1% for all data from 20 to 2.1 A, and the model geometry is excellent. The chromophore is non-native and is autocatalytically generated from the internal tripeptide Ser65-His66-Gly67. The final electron density maps indicate that the formation of the chromophore is complete, including 1,2 dehydration of His66 as indicated by the planarity of the chromophore. The chromophore is in the cis conformation, with no evidence for any substantial fraction of the trans configuration or uncyclized apoprotein, and is well-shielded from bulk solvent by the folded protein. These characteristics indicate that the machinery for production of the chromophore from a buried tripeptide unit is not only intact but also highly efficient in spite of a major change in chromophore chemical structure. Nevertheless, there are significant rearrangements in the hydrogen bond configuration around the chromophore as compared to wild-type, indicating flexibility of the active site. pH titration of the intact protein and the chromopeptide (pKa1 = 4.9 +/- 0.1, pKa2 = 12.0 +/- 0.1) suggests that the predominant form of the chromophore in the intact protein is electrically neutral. In contrast to the wild-type protein [Chattoraj, M., King, B. A., Bublitz, G. U., & Boxer, S. G. (1996) Proc. Natl. Acad. Sci. U.S.A., 8362-8367], femtosecond fluorescence up-conversion spectroscopy of the intact protein and a partially deuterated form strongly suggests that excited-state proton transfer is not coupled to fluorescence emission.     

How are psychotic symptoms perceived? A comparison between patients, relatives and the general public

OBJECTIVE: The aim of this study is to compare patients with schizophrenia with their relatives and the general public in their attitudes towards schizophrenic psychotic symptoms. METHOD: We used a case vignette depicting a person with typical schizophrenic psychotic symptoms and compared the attitudes of 44 inpatients and 47 outpatients with schizophrenia, 48 of their relatives and 43 members of the general public. We also compared the attitudes of patients with schizophrenia to their own symptoms and the symptoms described in the vignette. RESULTS: Subjects from the general public tended not to recognise psychotic symptoms as features of mental illness and tended not to consider drug treatment and hospitalisation as required. Sex, education level as well as previous contact with the mentally ill were found to be significant determinants of attitude. The levels of symptom awareness in patients with schizophrenia and their relatives are higher but still relatively low. In addition, we found that patients with schizophrenia who correctly appraised psychotic symptoms in another person were also aware of their own mental symptoms and need of treatment. CONCLUSIONS: The level of recognition of psychotic symptoms and awareness of a need for treatment are low in the general public, as well as in patients with schizophrenia and their relatives. These findings are discussed in relation to the assessment of insight in patients and a need for psychoeducational programs for each group.     

Current status of solvent/detergent-treated frozen plasma

DNA superhelical tension, an important feature of genomic organization, is known to affect the interactions of intercalating molecules with DNA. However, the effect of torsional tension on nonintercalative DNA-binding chemicals has received less attention. We demonstrate here that the enediyne calicheamicin gamma1I, a strand-breaking agent specific to the minor groove, causes approximately 50% more damage in negatively supercoiled plasmid DNA than in DNA with positive superhelicity. Furthermore, we show that the decrease in damage in positively supercoiled DNA is controlled at the level of thiol activation of the drug. Our results suggest that supercoiling may affect both the activity of nonintercalating genotoxins in vivo and the accessibility of glutathione and other small physiologic molecules to DNA-bound chemicals or reactions occurring in the grooves of DNA.     

Assessing of Natural Attenuation and Intrinsic Bioremediation Rates at a Petroleum-Hydrocarbon Spill Site: Laboratory and Field Studies

Natural attenuation is a passive remedial approach that depends upon natural processes to degrade and dissipate contaminants in soil and groundwater. Intrinsic bioremediation is believed to be the major process among the natural attenuation mechanisms that account for the reduction of contaminant concentrations. In this study, a mass flux approach was used to calculate the contaminant mass reduction at a petroleum-hydrocarbon spill site. The mass flux technique is a simplified mass balance procedure, which is accomplished using the differences in total contaminant mass flux across two cross sections of the contaminant plume. The mass flux calculation results show that up to 86% of the dissolved total benzene, toluene, ethylbenzene, and xylene (BTEX) isomers removal was observed via natural attenuation at this site. Evidence for the occurrence of natural attenuation was the decreased contaminant mass flux through the plume cross sections along the transport path and limited spreading of the BTEX plume. Evidences for the BTEX biodegradation include: (1) decreased BTEX concentrations along the transport path; (2) depletion of dissolved oxygen, nitrate, and sulfate; (3) production of dissolved ferrous iron, sulfide, methane, and CO2; (4) deceased pH in the spill source area and increased pH in iron-reducing area; (5) increased alkalinity and microbial populations; and (6) preferential removal of certain BTEX components along the transport path. The effectiveness of intrinsic bioremediation on BTEX removal was evaluated by the in situ tracer method. Results reveal that approximately 74% of the BTEX removal was due to the intrinsic biodegradation process. The first-order decay model was applied for the natural attenuation and intrinsic bioremediation rates calculation. Results show that the biodegradation capacity (34.5 mg/L) for BTEX was much higher than the detected contaminants within the plume. The calculated total BTEX first-order natural attenuation and intrinsic bioremediation rates were 0.025 and 0.017% 1/day, respectively. Results of polymerase chain reaction, denaturing gradient gel electrophoresis, and nucleotide sequence analysis reveal that some petroleum-hydrocarbon degraders (Flavobacterium capsulatum, Xanthobacter sp., Xanthobacter flavus, Xanthomonas codiaei, Pseudomonas boreopolis, Methylobacterium sp., Reichenowia pictae) might exist at this site, which might contribute to the BTEX biodegradation. Results suggest that the natural attenuation mechanisms can effectively contain the plume, and the mass flux method is useful in assessing the occurrence and efficiency of the natural attenuation and intrinsic bioremediation processes.