Queuine is incorporated into brain transfer RNA

Pig brain tRNA was assayed for the presence of queuosine in the first position of the anticodon for each of the Q-family of tRNAs (aspartyl, asparaginyl, histidyl and tyrosyl). The brain tRNA was aminoacylated with each of the four amino acids and the aminoacylated tRNA's analyzed by RPC-5 chromatography. The results of this study show that for all four tRNAs of the family, queuine is substituted for guanine in virtually 100% of the anticodons. Therefore, it can be concluded that queuine is able to cross the blood-brain barrier and that brain contains quanine-queuine tRNA transglycosylase, the enzyme responsible for the excision of guanine from the orginal transcipts of these tRNAs and insertion of queuine. The determination of whether the tRNA contained queuine was made from the elution profile of the RPC-5 chromatrograms and the results confirmed by a change in the RPC-5 elution profile when the tRNAs were reacted with BrCN or NaIO₄.     

Sterols and frost hardening of winter rape

Winter rape ( Brassica napus L., var. olifera cv. Górczanski) seedlings were exposed to hardening conditions and the content and composition of free sterols as well as the ratio of free sterols to total phospholipids were determined. There was a reduction in free sterol content in the leaves at the most advanced stage of hardening. The ratio of free sterol to total phospholipids was significantly reduced by hardening due to a decrease in the level of the former and an increase in that of the latter compounds. There was a negative correlation between this ratio and the temperature at which half of the seedlings died. Thus, adaptation of membranes to temperature takes place also at the level of sterol-phospholipid interactions. Exposing seedlings already hardened to freezing temperatures caused injury higher than 50%, and brought about a drastic increase in the level of free sterols and an elevation in the ratio of free sterols to phospholipids. The results are discussed in terms of a possible role of the molecular architecture of membranes in surviving at subzero temperatures.     

Hyperpolarization and intracellular acidification in Trichoderma viride as a response to illumination.

Using indirect methods based on uptake of [3H]tetraphenylphosphonium cation and [14C]benzoic acid by cells of the fungus Trichoderma viride we found that the illumination-induced transient hyperpolarization of the plasma membrane is followed immediately by a rapid temporary decrease in intracellular pH. Hyperpolarization and intracellular acidification were completely suppressed by 150 mM-KCl and by the K(+)-ionophore valinomycin. The light-induced acidification of the cytoplasm was not observed in the presence of the cytochrome respiratory chain inhibitors antimycin A and mucidin. Based on these results, we hypothesize that the hyperpolarization of the cells is the consequence of an efflux of K+ through a light-activated K(+)-channel in the plasma membrane. The loss of positive charge in the cytoplasm caused by this efflux of cations is counterbalanced by H+ originating from the light-activated mitochondrial respiratory chain.     

Photosensitizers and antioxidants: a way to new drugs?

Photodynamic therapy (PDT) is a relatively new modality of treatment of diseases involving uncontrolled cell proliferation. It is based on the production of reactive species upon illumination of a photosensitizer in the presence of oxygen. Antioxidants are primarily reducing agents prone to scavenge reactive species in one way or another. Their presence in photodynamic reactions usually reduces the efficacy of PDT. Some antioxidants like ascorbic acid, alpha-tocopherol or butyl-4-hydroxyanisole, however, when added to cells at adequate concentrations may enhance the photodamaging activity of PDT. The presence of transition metals and precise timing of antioxidant administration may also be important factors in increasing the efficacy of PDT. Antioxidant carrier sensitizers have been designed, synthesised and tested for their antibacterial PDT activity. The promising results raise the question whether the introduction of antioxidant moieties into sensitizer molecules would lead to the synthesis of highly effective new drugs.     

Versatile High Resolution Oligosaccharide Microarrays for Plant Glycobiology and Cell Wall Research

Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.