The effect of sample freezing on proton magic-angle spinning NMR spectra of biological tissue

Magic-angle spinning (MAS) has recently been shown to enhance spectral resolution in NMR examinations of intact biological tissue ex vivo. This work demonstrates that freezing certain tissue samples before examination by 1H MAS NMR can have a marked effect on their spectra. Spectra of rat kidney after freezing in liquid nitrogen, compared with spectra before freezing, showed a significant increase in signal intensities from alanine (>100%), glutamine (>40%), and glycine (>100%), and a decrease in signals assigned to lipids and other macromolecules. Some resonances--such as from leucine, valine, isoleucine, and aspartate--only became visible after freezing the tissue. These observations suggest that low temperature storage of tissue necropsies or biopsies might affect the results of a MAS NMR analysis, possibly resulting in the misinterpretation of metabolite changes to pathogen or disease effects.     

Health status and health care costs for publicly funded patients with schizophrenia started on clozapine

OBJECTIVE: The study examined the effect of clozapine treatment on the health care costs and health status of people with schizophrenia who are supported by public funds. METHODS: Thirty-three patients with schizophrenia hospitalized in a state facility were interviewed within one week of starting clozapine and six months later. Health status was assessed with four clinical rating scales measuring severity of psychopathology, negative symptoms, depression, and quality of life. Cost and health care utilization data were collected for the six months before and after initiation of clozapine. RESULTS: Only 52 percent of the subjects stayed on clozapine for six months. Subjects who continued on clozapine were more likely to be discharged within six months than those who did not continue. Six months after clozapine was started, health care costs showed a sayings of $11,464 per person, even after adjustment for pretreatment costs, and health status was improved. CONCLUSIONS: For subjects who continued on clozapine for six months, clozapine treatment was associated with reduced days of psychiatric hospital care, reduced overall costs despite increased outpatient treatment and residential costs, and improved health status.     

Determination of calcium-binding sites in rat brain calbindin D28K by electrospray ionization mass spectrometry

Calbindin D28K, a member of the troponin-C superfamily of calcium-binding proteins, contains six putative EF-hand domains. Calcium-binding studies of the protein by different groups of investigators have yielded discordant results with respect to the stoichiometry of calcium-binding. It has been suggested that the protein binds anywhere from 3-6 mol of calcium/mol of protein. We used negative ion electrospray ionization mass spectrometry in order to definitively determine the exact calcium-binding stoichiometry of calbindin D28K and two mutant forms of the protein, one lacking EF-hand 2 (delta2) and the other lacking EF-hands 2 and 6 (delta2,6). The full-length protein bound 4 mol of calcium/mol of protein, while both of the deletion mutants bound 3 mol of calcium. Since terbium has been used extensively as a probe for the determination of the calcium-binding stoichiometries of calcium-binding proteins, we also examined the binding of terbium to the three proteins under the same conditions. Full-length calbindin D28K bound 4 mol of terbium/mol of protein, while calbindin delta2 and delta2,6 each bound 3 mol. These results clearly show that calbindin D28K binds 4 mol of calcium/mol of protein and that terbium-binding stoichiometry is similar to that of calcium.     

Studies in metamerism using dichroic ladders

Dichroic ladders, comprising a set of samples and a floater dyed with different dyes, have been used to examine metameric pairs of coloured samples. Observers were asked to select the sample from the ladder which most closely matched the floater, the resulting pair being metameric. Whilst there was a good agreement between the visually and instrumentally selected matching ladder sample under illuminant D₅₅, consistent differences were found under illuminant A and TL84. An improvement in the level of agreement between the visual and the instrumentally determined matching ladder sample under illuminant A and TL84 was obtained if tristimulus values were computed from reflectance values using weighted ordinates interpolated to the 6d? and 16d? observer respectively.     

LPS-induced TNF-alpha release from and apoptosis in rat cardiomyocytes: obligatory role for CD14 in mediating the LPS response

The bacterial endotoxin lipopolysaccharide (LPS) contributes to the cardiovascular collapse and death observed in patients with sepsis. Because LPS has such profound effects on cardiac performance, we speculate that direct effects of LPS could be demonstrated on cardiomyocytes in culture, and that these direct effects are mediated by the LPS receptor, CD14. Accordingly, in this study, we provide evidence for CD14-dependent cardiotoxic effects of LPS including the LPS-stimulated secretion of tumor necrosis factor alpha (TNF-alpha) from cardiomyocytes. TNF-alpha is an inflammatory cytokine which is known for its negative inotropic effects on cardiac performance, but has not until recently been shown to be produced by cardiac cells. In this study, LPS was found to stimulate strongly in a dose-dependent manner the secretion of TNF-alpha from cultured adult rat cardiomyocytes. Further, LPS-induced TNF-alpha secretion was blocked by an inhibitor of TNF-alpha processing, metallomatrix protease inhibitor (TAPI). Molecular and immunological evidence demonstrated the presence of LPS receptors (CD14) on cardiomyocytes. Attenuated TNF-alpha secretion following PI-PLC treatment confirmed the functional importance of CD14 for LPS-mediated myocardial effects. Importantly, LPS also triggered apoptosis in cultured cardiomyocytes as quantified by single-cell gel electrophoresis of nuclei exhibiting DNA fragmentation patterns characteristic of apoptosis (i.e. cardiac comets). Apoptotic cell death was blocked by pre-incubation with the soluble TNF-alpha receptor fragment (TNFRII:Fc), suggesting that LPS-induced apoptosis was TNF-alpha-dependent and probably involved an autocrine function for the TNF-alpha whose secretion was under LPS control. The results of this study suggest that the cardiodepressant effects of LPS are dependent on CD14 signaling and may not only be due to acute negative inotropic effects of TNF-alpha but also may be complicated by TNF-alpha-induced apoptotic cell death which effectively reduces the number of working myocardial cells.     

Descending neural projections to the spinal cord in the channel catfish, Ictalurus punctatus

Retrograde transport of horseradish peroxidase was used to determine the descending projections to the spinal cord in an otophysan fish, the channel catfish, Ictalurus punctatus. The majority of cells projecting to the spinal cord are located in the reticular formation, which is organized into rhombomeric segments. Vestibulospinal neurons are located in the descending, magnocellular, and tangential octaval nuclei, as well as in the medial octavolateralis nucleus of the lateral line system. Cells in the facial lobe project to the spinal cord. Additionally, axons of cells of the trigeminal system and the nucleus of the lateral lemniscus project caudally into the spinal cord. In the midbrain, descending spinal projections arise from cells of the medial longitudinal fasciculus and the red nucleus. More rostrally, cells of the ventrolateral thalamus, dorsal periventricular hypothalamus, central pretectal and magnocellular preoptic nuclei also project to the cord. The results of this study indicate that there are a number of homologies in the descending systems of bony fishes and other vertebrate taxa, including tetrapods. We also provide further evidence that a red nucleus is present in the brains of bony fishes and is therefore a primitive vertebrate character antedating the evolution of tetrapods.     

Causal attributions of hyperactive children: Implications for teaching strategies and self-control.

The combined influence of attribution and self-control training on the short- and long-term maintenance of strategic behavior, impulsivity, and beliefs about self-efficacy was assessed in 77 underachieving, hyperactive children. During 4 sessions, children in a self-control condition received self-management training and instructions in the use of interrogative and clustering rehearsal strategies. Children in a self-control plus attribution condition received the same instructions plus training designed to enhance general and program-specific beliefs about the importance of effort in improving performance. Children in a control condition were given strategy training but received no self-control or attribution instructions. Analysis of short-term treatment effects showed that children who received attribution training used more complex strategies, demonstrated higher personal causality scores endorsing effort, and displayed reduced impulsivity. Ten months following training, children in the self-control plus attribution condition persisted in their use of acquired strategies, maintained beliefs about the importance of effort, and displayed more mature memory knowledge. Severely hyperactive children, who had received the attributional boost, showed decreased hyperactivity in the classroom and improved self-control. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Deletion of the H19 differentially methylated domain results in loss of imprinted expression of H19 and Igf2

Differentially methylated sequences associated with imprinted genes are proposed to control genomic imprinting. A 2-kb region located 5' to the imprinted mouse H19 gene is hypermethylated on the inactive paternal allele throughout development. To determine whether this differentially methylated domain (DMD) is required for imprinted expression at the endogenous locus, we have generated mice harboring a 1.6-kb targeted deletion of the DMD and assayed for allelic expression of H19 and the linked, oppositely imprinted Igf2 gene. H19 is activated and Igf2 expression is reduced when the DMD deletion is paternally inherited; conversely, upon maternal transmission of the mutation, H19 expression is reduced and Igf2 is activated. Consistent with the DMD's hypothesized role of setting up the methylation imprint, the mutation also perturbs allele-specific methylation of the remaining H19 sequences. In conclusion, these experiments show that the H19 hypermethylated 5' flanking sequences are required to silence paternally derived H19. Additionally, these experiments demonstrate a novel role for the DMD on the maternal chromosome where it is required for the maximal expression of H19 and the silencing of Igf2. Thus, the H19 differentially methylated sequences are required for both H19 and Igf2 imprinting.