Apoprotein synthesis by human duodenojejunal mucosa.

We tested whether human duodenojejunal mucosa is able to synthesize apoproteins from amino acid precursors because lipoprotein-like particles are visualized by electron microscopy in human absorptive cells and because apoproteins are synthesized by the perfused rat intestine. Duodenojejunal biopsies from 21 normal fasting volunteers were incubated with L-[U-14C]leucine; 15.6 +/- 3.3 nmoles of the [14C]leucine was incorporated into protein by 100 mg wet weight of biopsies during an incubation of 2 hr. No [14C]leucine was incorporated by boiled biopsies. Homogenates of incubated biopsies were fractionated by ultracentrifugation: 43 +/- 2% of the incorporated 14C as found in the d less than 1.006 fraction; 3.2 +/- 0.6%, 8.1 +/- 1.0% and 48 +/- 2% were found to be associated with the d = 1.006 to 1.063, d = 1.063 to 1.25, and d greater than 1.25 fractions, respectively. The specific activity in the d less than 1.006 fraction was 5.6 times greater than that in the other fractions. Of the 14C incorporated into the d less than 1.006 fraction, 10.8, 7.2, and 7.1% were specifically precipitated by rabbit antihuman apoproteins A-I, A-II, and B, respectively. Of the 14C incorporated into the d = 1.006 to 1.063 fraction, 11.3 4.1, and 8.5% were specifically precipitated by rabbit antihuman apoprotein A-I, A-II, and B, respectively. Approximately 5% of the 14C incorporated by the d = 1.063 to 1.25 fractions were precipitated by rabbit antihuman apoprotein A-I or A-II. None of the d less than 1.25 fractions precipitated a significant amount of radioactivity with rabit antihuman apoprotein C-II, or antiarginine-rich apoprotein. None of the antibodies precipitated radioactivity from the d greater than 1.25 fraction. These experiments suggest that human duodenojejunal mucosa is able to synthesize in vitro apoproteins A-I, A-II, and B from amino acid precursors. The specificity of the immunoprecipitates was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

Spinal cord injury and protection

Subsequent to traumatic injury of the spinal cord, a series of pathophysiological events occurs in the injured tissue that leads to tissue destruction and paraplegia. These include hemorrhagic necrosis, ischemia, edema, inflammation, neuronophagia, loss of Ca2+ from the extracellular space, and loss of K+ from the intracellular space. In addition, there is trauma-initiated lipid peroxidation and hydrolysis in cellular membranes. Both lipid peroxidation and hydrolysis can damage cells directly; hydrolysis also results in the formation of the biologically active prostaglandins and leukotrienes (eicosanoids). The time course of membrane lipid alterations seen in studies of antioxidant interventions suggests that posttraumatic ischemia, edema, inflammation, and ionic fluxes are the result of extensive membrane peroxidative reactions and lipolysis that produce vasoactive and chemotactic eicosanoids. A diverse group of compounds has been shown to be effective in ameliorating spinal cord injury in experimental animals. These include the synthetic glucocorticoid methylprednisolone sodium succinate (MPSS); the antioxidants vitamin E, selenium, and dimethyl sulfoxide (DMSO); the opiate antagonist naloxone; and thyrotropin-releasing hormone (TRH). With the exception of TRH, all of these agents have demonstrable antioxidant and/or anti-lipid-hydrolysis properties. Thus the effectiveness of these substances may lie in their ability to quench membrane peroxidative reactions or to inhibit the release of fatty acids from membrane phospholipids, or both. Whatever the mode of action, early administration appears to be a requirement for maximum effectiveness.     

Age-dependent neurological phenotypes in a mouse model of PRRT2-related diseases

Paroxysmal kinesigenic dyskinesia is an episodic movement disorder caused by dominant mutations in the proline-rich transmembrane protein PRRT2, with onset in childhood and typically with improvement or resolution by middle age. Mutations in the same gene may also cause benign infantile seizures, which begin in the first year of life and typically remit by the age of 2 years. Many details of PRRT2 function at the synapse, and the effects of mutations on neuronal excitability in the pathophysiology of epilepsy and dyskinesia, have emerged through the work of several groups over the last decade. However, the age dependence of the phenotypes has not been explored in detail in transgenic models. Here, we report our findings in heterozygous and homozygous Prrt2 knockout mice that recapitulate the age dependence of dyskinesia seen in the human disease. We show that Prrt2 deletion reduces the levels of synaptic proteins in a dose-dependent manner that is most pronounced at postnatal day 5 (P5), attenuates at P60, and disappears by P180. In a test for foot slippage while crossing a balance beam, transient loss of coordination was most pronounced at P60 and less prominent at age extremes. Slower traverse time was noted in homozygous knockout mice only, consistent with the ataxia seen in rare individuals with biallelic loss of function mutations in Prrt2. We thus identify three age-dependent phenotypic windows in the mouse model, which recapitulate the pattern seen in humans with PRRT2-related diseases.

     

Pathways to earned-security: The role of alternative support figures

This study explored the kinds of relationship experiences associated with earned-security, i.e., the extent to which mothers who report early negative relationship histories with their parents are later able to form a secure working model of attachment (indicated by the ability to speak clearly and coherently about these histories). Mothers from a low-risk sample (N = 121) expecting their first child completed the Adult Attachment Interview (AAI), which was used to assess earned-security retrospectively using the stringent definition recommended by Main and Hesse (Hesse, 2008 Hesse, E. 2008. “The Adult Attachment Interview: Protocol, method of analysis, and empirical studies”. In The handbook of attachment: Theory, research and clinical applications, Edited by: Cassidy, J. and Shaver, P. R. 552–598. New York, NY: The Guilford Press.  [Google Scholar]; Main, Goldwyn, & Hesse, 2002 Main, M., Goldwyn, R. and Hesse, E. 2002/2008. Adult attachment scoring and classification system, Unpublished scoring manual, University of California at Berkeley.  [Google Scholar]), as well as to identify alternative support figures. Participants also completed self-report measures of depressive symptomatology, questionnaires concerning their experiences in therapy, and later, when their babies were 12 to 15 months old, the Strange Situation procedure. Sixteen mothers were classified as earned-secure (25% of those classified as secure-autonomous and 13% of the whole sample). Women who were earned-secure (vs. insecure and continuous-secure) reported significantly higher levels of emotional support, but not instrumental support, from alternative support figures. They also spent more time in therapy than did insecure and continuous-secure women and were more likely to form secure attachments with their infants than insecure women. These findings were obtained even after controlling for depressive symptoms.     

Bromhexine in severe asthma.

Fourteen patients with acute severe asthma necessitating hospital admission were all treated with a standard therapeutic regimen, and in addition received either bromhexine or placebo (initially intravenously and subsequently orally) double-blind. We were unable to show any significant improvement in the rate of recovery of the bromhexine-treated group, in terms of either arterial blood gases or change in peak expiratory flow rates.     

Experimental design for stable genetic manipulation in mammalian cell lines: lentivirus and alternatives

The use of third‐generation lentiviral vectors is now commonplace in most areas of basic biology. These systems provide a fast, efficient means for modulating gene expression, but experimental design needs to be carefully considered to minimize potential artefacts arising from off‐target effects and other confounding factors. This review offers a starting point for those new to lentiviral‐based vector systems, addressing the main issues involved with the use of lentiviral systems in vitro and outlines considerations which should be taken into account during experimental design. Factors such as selecting an appropriate system and controls, and practical titration of viral transduction are important considerations for experimental design. We also briefly describe some of the more recent advances in genome editing technology. TALENs and CRISPRs offer an alternative to lentivirus, providing endogenous gene editing with reduced off‐target effects often at the expense of efficiency.