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.     

The plasma membrane of Streptococcus cremoris: isolation and partial characterization

Plasma membrane was isolated from Streptococcus cremoris using mutanolysin from a streptomycete as the cell wall-degrading enzyme and phenylmethylsulfonyl fluoride as protease inhibitor. The specific activity of membrane-bound enzyme, adenosine triphosphatase (ATPase), was 4 μmol/mg protein per min, which was 5–10 times higher than the activity found in other fractions obtained during the isolation procedure. The number of polypeptides in the plasma membrane was approximately 50 with molecular weights 13 500–100 000, minor changes in the polypeptide pattern were observed when the plasma membrane was isolated without a protease inhibitor. The chemical composition of the membrane preparation was 49.7% protein, 21.9% lipid, 5.1% aminosugars, 17.3% RNA and 0.03% DNA. Electron microscopic examination confirmed the membrane to be practically devoid of cell wall components. Our results indicate that the membrane integrity is well retained and therefore the membrane preparation is suitable for detailed studies on vectorial metabolism and its enzymes, e.g. ATPase.     

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.     

Hepatocyte Growth Factor Activator Inhibitor-1 Is Induced by Bone Morphogenetic Proteins and Regulates Proliferation and Cell Fate of Neural Progenitor Cells

Background

Neural progenitor cells (NPCs) in the developing neuroepithelium are regulated by intrinsic and extrinsic factors. There is evidence that NPCs form a self-supporting niche for cell maintenance and proliferation. However, molecular interactions and cell-cell contacts and the microenvironment within the neuroepithelium are largely unknown. We hypothesized that cellular proteases especially those associated with the cell surface of NPCs play a role in regulation of progenitor cells in the brain.

Methodology/Principal Findings

In this work, we show that NPCs, isolated from striatal anlage of developing rat brain, express hepatocyte growth factor activator inhibitor-1 and -2 (HAI-1 and HAI-2) that are cell surface-linked serine protease inhibitors. In addition, radial glia cells derived from mouse embryonic stem cells also express HAI-1 and HAI-2. To study the functional significance of HAI-1 and HAI-2 in progenitor cells, we modulated their levels using expression plasmids or silencing RNA (siRNA) transfected into the NPCs. Data showed that overexpression of HAI-1 or HAI-2 decreased cell proliferation of cultured NPCs, whilst their siRNAs had opposite effects. HAI-1 also influenced NPC differentiation by increasing the number of glial fibrillary acidic protein (GFAP) expressing cells in the culture. Expression of HAI-1 in vivo decreased cell proliferation in developing neuroepithelium in E15 old animals and promoted astrocyte cell differentiation in neonatal animals. Studying the regulation of HAI-1, we observed that Bone morphogenetic protein-2 (BMP-2) and BMP-4 increased HAI-1 levels in the NPCs. Experiments using HAI-1-siRNA showed that these BMPs act on the NPCs partly in a HAI-1-dependent manner.

Conclusions

This study shows that the cell-surface serine protease inhibitors, HAI-1 and HAI-2 influence proliferation and cell fate of NPCs and their expression levels are linked to BMP signaling. Modulation of the levels and actions of HAI-1 in NPCs may be of a potential value in stem cell therapies in various brain diseases.     

Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes

We describe here a new method for highly efficient detection of microRNAs by northern blot analysis using LNA (locked nucleic acid)-modified oligonucleotides. In order to exploit the improved hybridization properties of LNA with their target RNA molecules, we designed several LNA-modified oligonucleotide probes for detection of different microRNAs in animals and plants. By modifying DNA oligonucleotides with LNAs using a design, in which every third nucleotide position was substituted by LNA, we could use the probes in northern blot analysis employing standard end-labelling techniques and hybridization conditions. The sensitivity in detecting mature microRNAs by northern blots was increased by at least 10-fold compared to DNA probes, while simultaneously being highly specific, as demonstrated by the use of different single and double mismatched LNA probes. Besides being highly efficient as northern probes, the same LNA-modified oligonucleotide probes would also be useful for miRNA in situ hybridization and miRNA expression profiling by LNA oligonucleotide microarrays.     

Functional cloning of an endo-arabinanase from Aspergillus aculeatus and its heterologous expression in A. or oryzae and tobacco

Functional cloning in yeast has been used to isolate full-length cDNAs encoding an endo-alpha-1,5-L-arabinanase from the filamentous fungus Aspergillus aculeatus. Screening of a cDNA library constructed in a yeast expression vector for transformants that hydrolysed AZCL-arabinan identified 44 Saccharomyces cerevisiae clones all harbouring the same arabinanase-encoding cDNA. The cloned cDNA was expressed in A. oryzae and the recombinant enzyme was purified and characterized. The mode of action of the enzyme was studied by analysis of the digestion pattern towards debranched arabinan. The digestion profile obtained strongly suggests that the enzyme is an endo-arabinanase. In addition, the feasibility using Nicotiana tabacum as an alternative host for arabinanase expression was investigated.