An initial top-down proteomic analysis of the standard cuprizone mouse model of multiple sclerosis

An initial proteomic analysis of the cuprizone mouse model to characterise the breadth of toxicity by assessing cortex, skeletal muscle, spleen and peripheral blood mononuclear cells. Cuprizone treated vs. control mice for an initial characterisation. Select tissues from each group were pooled, analysed in triplicate using two-dimensional gel electrophoresis (2DE) and deep imaging and altered protein species identified using liquid chromatography tandem mass spectrometry (LC/MS/MS). Forty-three proteins were found to be uniquely detectable or undetectable in the cuprizone treatment group across the tissues analysed. Protein species identified in the cortex may potentially be linked to axonal damage in this model, and those in the spleen and peripheral blood mononuclear cells to the minimal peripheral immune cell infiltration into the central nervous system during cuprizone mediated demyelination. Primary oligodendrocytosis has been observed in type III lesions in multiple sclerosis. However, the underlying mechanisms are poorly understood. Cuprizone treatment results in oligodendrocyte apoptosis and secondary demyelination. This initial analysis identified proteins likely related to axonal damage; these may link primary oligodendrocytosis and secondary axonal damage. Furthermore, this appears to be the first study of the cuprizone model to also identify alterations in the proteomes of skeletal muscle, spleen and peripheral blood mononuclear cells. Notably, protein disulphide isomerase was not detected in the cuprizone cohort; its absence has been linked to reduced major histocompatibility class I assembly and reduced antigen presentation. Overall, the results suggest that, like experimental autoimmune encephalomyelitis, results from the standard cuprizone model should be carefully considered relative to clinical multiple sclerosis.     

Non-Selective Calcium Channel Blocker Bepridil Decreases Secondary Pathology in Mice after Photothrombotic Cortical Lesion

Experimental studies have identified a complex link between neurodegeneration, β-amyloid (Aβ) and calcium homeostasis. Here we asked whether early phase β-amyloid pathology in transgenic hAPP_SL mice exaggerates the ischemic lesion and remote secondary pathology in the thalamus, and whether a non-selective calcium channel blocker reduces these pathologies. Transgenic hAPP_SL (n = 33) and non-transgenic (n = 30) male mice (4–5 months) were subjected to unilateral cortical photothrombosis and treated with the non-selective calcium channel blocker bepridil (50 mg/kg, p.o., once a day) or vehicle for 28 days, starting administration 2 days after the operation. Animals were then perfused for histological analysis of infarct size, Aβ and calcium accumulation in the thalamus. Cortical photothrombosis resulted in a small infarct, which was associated with atypical Aβ and calcium accumulation in the ipsilateral thalamus. Transgenic mice had significantly smaller infarct volumes than non-transgenic littermates (P<0.05) and ischemia-induced rodent Aβ accumulation in the thalamus was lower in transgenic mice compared to non-transgenic mice (P<0.01). Bepridil decreased calcium load in the thalamus (P<0.01). The present data suggest less pronounced primary and secondary pathology in hAPP_SL transgenic mice after ischemic cortical injury. Bepridil particularly decreased calcium pathology in the thalamus following ischemia.     

Effects on pregnancy in mice of passive immunization against ovine LH and human chorionic gonadotrophin

Mice given daily i.p. injections of immunoglobulins against ovine LH on Days 3-7 of pregnancy were devoid of implantation sites on Day 8 whereas mice treated with antibodies to hCG had embryos of normal number and appearance on Day 8. These antibody treatments reduced the mean +/- s.d. serum progesterone concentrations from 65.4 +/- 15.3 ng/ml (control globulins) to 8.6 +/- 4.9 ng/ml (anti-LH) and 9.2 +/- 3.1 ng/ml (anti-hCG) on Day 8 and had no differential effect on serum oestrogen levels on Day 4. However, the mice treated with anti-hCG did not litter; resorption of the embryos took place between Days 10 and 14 of pregnancy. Indirect immunofluorescence and quantitative immunoenzymic assays showed the presence of anti-ovine LH and anti-hCG reacting antigens in the mouse feto-placental unit. On Day 6, the values of reacting antigens (mean +/- s.d. absorbance units/10 micron section of embryo) were 0.050 +/- 0.002 with control globulins, 0.059 +/- 0.002 with anti-hCG-Ig and 0.196 +/- 0.018 with anti-LH-Ig; the corresponding values on Day 12 were 0.075 +/- 0.009, 0.402 +/- 0.02 and 0.416 +/- 0.015. The quantitative disposition of the reacting antigens to the two types of anti-gonadotrophins seems to bear a temporal relationship to their respective antifertility action. The pregnancy terminating action of immunoglobulins to ovine LH (Days 6, 7 & 8) and hCG (Days 8, 9 & 10) was counteracted by administration of 2 mg medroxyprogesterone acetate on Days 6, 9 and 12, indicating the importance of progesterone in the maintenance of pregnancy in the mouse.     

Lymphoid cell subclasses in rejecting renal allograft in the rat

We have quantitated the frequency of lymphoid cell subsets in rejecting renal allografts and in the spleen of the allograft recipient during drug-unmodified rejection in the rat. The number of inflammatory (white) cells in the graft was approximately similar to the number of white cells responding to the allograft in the recipient spleen. The inflammatory population of the graft consisted of lymphoid cells and mononuclear phagocytes, with increasing numbers of macrophages toward the end of rejection. Analysis of allograft cellular dispersates with monoclonal antibodies directed to the lymphoid cell subsets demonstrated that although the majority of allograft-infiltrating lymphocytes were T cells, a sizable B-cell proliferation and immunoglobulin synthesis was associated with the inflammatory response of rejection. Within the T-cell subset, the T suppressor/killer cells predominated in the graft whereas the predominant lymphoid cell subset responding to the allograft in the recipient spleen was the T helper cell.     

‘Leaf tip hooding’ — an induced mutation inN. Tabacum,var. Delcrest

A leaf tip hooded mutant was isolated in the progenies of the irradiated seeds of a commercial tobacco variety, namely Delcrest. The mutant character was determined as monogenic recessive. Along with leaf tip hooding, certain other characters such as increase in size of leaves, number and clasping arrangement of leaves on stem, and delayed flowering, were found to be closely associated and this was attributed to the pleiotropic effect of the leaf tip hooding gene.     

Fluidity of detergent micelles plays an important role in muscarinic receptor solubilization

In order to find a suitable reagent for extracting the muscarinic receptor from rat brain membranes 14 different detergents were tested. Only the plant glycoside digitonin efficiently solubilized the receptor protein in its native form. At the same time microviscosity of detergent micelles was determined by measuring the fluorescence polarization of a hydrophobic fluorescent probe diphenylhexatriene incorporated into the micelles. In the case of digitonin the polarization value was close to the corresponding value obtained for rat brain membrane fragments, while for the other detergents studied it remained considerably lower. The results obtained indicate that the fluidity of detergent micelles may play an important role in retaining the active conformation of the solubilized muscarinic receptor.     

Ter-Seq: A high-throughput method to stabilize transient ternary complexes and measure associated kinetics

Regulation of biological processes by proteins often involves the formation of transient, multimeric complexes whose characterization is mechanistically important but challenging. The bacterial toxin CcdB binds and poisons DNA Gyrase. The corresponding antitoxin CcdA extracts CcdB from its complex with Gyrase through the formation of a transient ternary complex, thus rejuvenating Gyrase. We describe a high throughput methodology called Ter-Seq to stabilize probable ternary complexes and measure associated kinetics using the CcdA-CcdB-GyrA14 ternary complex as a model system. The method involves screening a yeast surface display (YSD) saturation mutagenesis library of one partner (CcdB) for mutants that show enhanced ternary complex formation. We also isolated CcdB mutants that were either resistant or sensitive to rejuvenation, and used surface plasmon resonance (SPR) with purified proteins to validate the kinetics measured using the surface display. Positions, where CcdB mutations lead to slower rejuvenation rates, are largely involved in CcdA-binding, though there were several notable exceptions suggesting allostery. Mutations at these positions reduce the affinity towards CcdA, thereby slowing down the rejuvenation process. Mutations at GyrA14-interacting positions significantly enhanced rejuvenation rates, either due to reduced affinity or complete loss of CcdB binding to GyrA14. We examined the effect of different parameters (CcdA affinity, GyrA14 affinity, surface accessibilities, evolutionary conservation) on the rate of rejuvenation. Finally, we further validated the Ter-Seq results by monitoring the kinetics of ternary complex formation for individual CcdB mutants in solution by fluorescence resonance energy transfer (FRET) studies.     

Effects of long-term open-air exposure to fluoride,nitrogen compounds and SO2 on visible symptoms,pollutant accumulation and ultrastructure of Scots pine and Norway spruce seedlings

 Effects of SO₂, aqueous fluoride (NaF) and a solution of nitrogen compounds (NH₄NO₃) on the visible symptoms, pollutant accumulation and ultrastructure of Scots pine (Pinus sylvestris L.) and Norway spruce [Picea abies (L.) Karst.] seedlings were studied in an open-air experiment lasting for 3 consecutive years. Visible injury symptoms were most pronounced in combination exposures and whenever F was applied. Visible symptoms correlated well with needle pollutant concentrations. Exposure to NaF increased needle F contents particularly when F was applied with SO₂ or NH₄NO₃. This suggests that a reduction in N or SO₂ emissions, in F polluted areas, could improve the condition of conifers via decreased accumulation of phytotoxic F in the needles. Norway spruce needles accumulated 2 – 10 times as much S and F as those of Scots pine. Microscopic observations showed various changes in the needle mesophyll cell ultrastructure. In both species, exposure to SO₂ increased significantly the amount of cytoplasmic vacuoles, suggesting detoxification of excess sulphate or low pH. F treatments resulted in a significant enlargement of plastoglobuli in Scots pine and a darkening of plastoglobuli in Norway spruce. All exposures enhanced the accumulation of lipid bodies. An increased portion of translucent plastoglobuli was most pronounced in N treatments. Many of the ultrastructural changes and visible symptoms appeared only as number of years exposed increased, indicating that long-term experiments are needed. Both visible symptoms and ultrastructural changes pointed to the more pronounced sensitivity of Norway spruce compared to Scots pine. Ultrastructural results mostly supported earlier qualitative observations of F, N and SO₂ effects on needle mesophyll cell ultrastructure. However, no reduction of thylakoids in SO₂ containing exposure or curling of thylakoids in F exposure could be detected in the present study. Received: 5 December 1994 / Accepted: 28 April 1995     

Production of extracellular lipase by the hexachlorocyclohexane utilizing Pseudomonas strain Ptm+

Extracelluar lipase activity was detected in a culture of Pseudomonas strain Ptm⁺ growing on hexachlorocyclohexane. Lipase activity was associated with growth of the bacterium and reached a peak at early idiophase (22 h). Following the lipase activity, bioemulsifier was produced, with maximum activity at mid-idiophase (32 h). The extracellular lipase is probably involved in the extracellular synthesis of the bioemulsifier in the culture broth.     

Synthesis of carbamyl and ether analogs of phosphatidylcholines

A complete synthesis of 1-O-hexadecyl-2-O-N-(heptadec-8-cis-enyl)carbamyl-sn-glycero-3-Phosphocholine, a novel analog of phosphatidylcholine, has been described. Each step is simple to perform and gives the desired products in high yield. Also, some of the intermediates formed during the synthesis have been efficiently utilized to prepare 1-O-hexadecyl-2-O-oleyl-sn-glycero-3-phosphocholine, 1-O-hexadecyl-2-oleoyl-sn-glycero-3-phosphochloine and 3-O-hexadecyl-2-oeloyl-sn-glycero-1-phosphocholine. These phosphatidylcholine (PC) analogs are useful for studying the possible role of phospholipases in the capture and lyses of liposomes in vivo.