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.     

Proton electrochemical potential of the inner mitochondrial membrane in isolated perfused rat hearts, as measured by exogenous probes

The membrane potential (delta psi) and delta pH of the inner mitochondrial membrane were studied in isolated perfused rat hearts using exogenous labelled probes and tissue fractionation in non-aqueous media. The mitochondrial delta psi, measured by means of the subcellular distribution of [3H]triphenylmethylphosphonium (TPMP+), was 125 +/- 7 mV (negative inside) in hearts beating at 5 Hz and 150 +/- 3 mV (negative inside) in hearts beating at 1.5 Hz. The mitochondrial membrane delta pH, measured by means of the subcellular distribution of low concentrations of [1-14C]propionate, was 0.63 +/- 0.06 pH units (alkaline inside) in hearts beating at 5 Hz and 0.53 +/- 0.12 pH units (alkaline inside) in hearts beating at 1.5 Hz. The implication of proton and electron gradients in the regulation of cellular respiration is discussed. In combination with previous evidence on adenylate distribution in the isolated perfused rat heart, the results indicate that the mitochondrial electrogenic adenylate translocator is in near equilibrium with delta psi.     

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.     

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.     

Secretion of a heat-stable fungalβ-glucanase from transgenic,suspension-cultured barley cells

Transgenic plant cell cultures have a potential for production and secretion of important proteins and peptides. To assess the possibilities of using a stable barley suspension culture for secretion of heterologous proteins in active form, we expressed the cDNA of the thermostable-glucanase (EGI) ofTrichoderma reesei in barley suspension cells. The cDNA coding for EGI and its signal sequence was placed under the control of the CaMV 35S promoter and the construction was transferred to the cells by particle bombardment. Stably transformed lines were obtained by selecting for a cotransformed antibiotic resistance marker. The expression of EGI cDNA led to accumulation of EGI in the culture medium, as shown by analysis with EGI-specific antibodies. Enzymatic assays confirmed that the EGI secreted by the suspension cells retained its activity and thermostable character. Furthermore, it was shown that the enzyme produced by the transgenic suspension culture could be used for degradation of soluble-glucans during mashing.     

Fertile transgenic barley by particle bombardment of immature embryos

Transgenic, fertile barley (Hordeum vulgare L.) from the Finnish elite cultivar Kymppi was obtained by particle bombardment of immature embryos. Immature embryos were bombarded to the embryonic axis side and grown to plants without selection. Neomycin phosphotransferase II (NPTII) activity was screened in small plantlets. One out of a total of 227 plants expressed the transferred nptII gene. This plant has until now produced 98 fertile spikes (T₀), and four of the 90 T₀ spikes analyzed to date contained the nptII gene. These shoots were further analyzed and they expressed the transferred gene. From green grains, embryos were isolated and grown to plantlets (T₁). The four transgenic shoots of Toivo (the T₀ plant) produced 25 plantlets as T₁ progeny. Altogether fifteen of these T₁ plants carried the transferred nptII gene as detected with the PCR technique, fourteen of which expressed the nptII gene. The integration and inheritance of the transferred nptII gene was confirmed by Southern blot hybridization. Although present as several copies, the transferred gene was inherited as a single Mendelian locus into the T₂ progeny.     

1H Nuclear Magnetic Resonance Spectroscopy Study of Cerebral Glutamate in an Ex Vivo Brain Preparation of Guinea Pig

Abstract: Cerebral glutamate was monitored in a superfused cerebral cortical preparation by ¹H NMR spectroscopy using a semiselective spin-echo sequence N -acetyl aspartate (NAA) as an internal concentration reference. During controlled metabolic conditions, the cerebral ¹H NMR-detected glutamate-to-NAA ratio was ∼ 20–30% lower than expected from the ratio of neutralized perchloric acid extracts of the preparations. Inhibition of respiration in the presence of glucose did not change the ¹H NMR glutamate-to-NAA ratio in brain slice preparation. In contrast, either complete depletion of ATP during cyanide poisoning together with 0 m M glucose, anoxia in the absence of glucose, or treatment with nigericin or with a protonophore, carbonyl cyanide- m -fluorophenylhydrazone, increased ¹H NMR-detected glutamate/NAA in the cerebral preparations without a change in the relative and absolute concentration ratios determined from the tissue acid extracts. Spin-spin relaxation times of glutamate and NAA peaks in anoxic slices were 749 ± 89 and 729 ± 94 ms, respectively, and thus, the portion of glutamate that could not be detected by ¹H NMR was quantified in absolute terms. It was calculated that an increase in the glutamate-to-NAA ratio from 0.55 ± 0.02 to 0.67 ± 0.02 during aglycemic anoxia corresponded to some 6 mmol/kg of tissue dry weight of glutamate from the total concentration of 28 mmol/kg dry weight. It is suggested that this 22% of total glutamate pool is present in a noncytoplasmic compartment during controlled metabolic state.     

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.     

The impact of a salinity barrier on the partitioning of heavy metals in sediments of a tropical backwater system

Abstract

Concentrations of heavy metals (Cd, Co, Cr, Cu and Fe) in surface sediments and their partitioning behaviour between exchangeable, reducible (Fe-Mn oxide bound) and organic/residual phases of the sediments in a typical backwater system of Kerala, viz. the southern upstream part of Cochin Estuarine System (South India), have been studied. Spatial and temporal variations of trace metals are discussed with special reference to pH, dissolved oxygen, salinity, organic carbon and texture of sediment. Metal concentrations in the tide gated part of the estuary were found to be significantly higher when compared to metal concentrations reported from the unrestricted part of the Cochin estuarine system and also those from many of the unpolluted estuaries worldwide. The higher levels of heavy metals in the study area and their characteristic distribution and partitioning behaviour in the surficial sediments were attributed to the presence of a salinity barrier across the backwater system and also by the massive use of pesticides and chemical fertilizers in the vast area of agricultural land near the backwater system.