Recent approaches to probe functional groups in ribonuclease P RNA by modification interference

Modification interference is a powerful method to identify important functional groups in RNA molecules. We review here recent developments of techniques to screen for chemical modifications that interfere with (i) binding of(pre-)tRNA to bacterial RNase P RNA or (ii) pre-tRNA cleavage by this ribozyme. For example, two studies have analyzed positions at which a substitution of sulfur for thepro-Rp oxygen affects tRNA binding [1] or catalysis [2]. The results emphasize the functional key role of a central core element present in all known RNase P RNA subunits. The four sulfur substitutions identified in one study [2] to inhibit the catalytic step also interfered with binding of tRNA toE. coli RNase P RNA [1]. This suggests that losses in binding energy due to the modification at these positions affect the enzyme-substrate and the enzyme-transition state complex. In addition, the two studies have revealed, for the first time, sites of direct metal ion coordination in RNase P RNA. The potentials, limitations and interpretational ambiguities of modification interference experiments as well as factors influencing their outcome are discussed.Abbreviations nt nucleotide(s) - PAGE polyacrylamide gel electrophoresis     

Expression of the plant sulphite reductase in cell suspension cultures from Catharanthus roseus L.

Sulphur-heterotrophic growth exhibited a dual response to the expression of sulphate-assimilating enzymes. The level of ATP-sulphurylase (EC 2.7.7.4) appeared repressed while sulphite reductase (EC 1.8.7.1) and O-acetyl-l-serine sulphhydrylase (EC 4.2.99.8) were derepressed and coordinated in their occurrence. The capability of the cells to reduce adenylylphosphosulphate or 3-phospho adenylylphosphosulphate to cysteine coincided with the activity of sulphite reductase. The expression of these reducing steps lacked correlation with the regulation of ATP-sulphurylase.Abbreviations APS adenylylphosphosulphate - MVH reduced methylviologen - OAS O-acetyl-l-serine - PAPS 3-phospho adenylylphosulphate     

An improved timm sulphide silver method for light and electron microscopic localization of heavy metals in biological tissues

Summary Modifications of the Timm sulphide silver method for the demonstration of heavy metals are described.To improve the structural preservation of the tissues perfusion with a glutaraldehyde fixative is employed before perfusion with the sodium sulphide solution. For the subsequent staining for light and electron microscopy, procedures for plastic embedding, paraffin embedding and cryostat sectioning are presented. Examples from several tissues are shown, including the pituitary, pancreas, intestine, tongue, kidney, testis and brain. The staining of autolytic, postmortal human brain tissue is demonstrated.     

Mitochondrial calcium uptake during infection of chicken embryo cells with Semliki Forest virus.

The key role of the mitochondria in the regulation of cellular Ca2+ led to a study of mitochondrial Ca2+ uptake during the infection of chicken embryo cells with Semliki Forest virus. Mitochondrial Ca2+ uptake was stimulated during the first 5 h of infection but declined later in infection. The early stimulation suggests an increase of cytoplasmic ionized Ca2+, whereas the later decrease indicates mitochondrial injury. This functional deterioration was correlated with an increase of the permeability of the inner mitochondrial membrane. Polarographic experiments showed that electron transport is impaired, whereas transduction of energy to Ca2+ uptake is intact.     

Alveld-Producing Saponins

Stabursvik (1959) described the saponin fraction of Narthecium ossifragum as a sarsasapogenin glycoside with the structure arabinosegalactose-xylose-glucose-sarsasapogenin. In a renewed study of the phototoxic lamb disease alveld, in which this saponin has been implicated (Ender 1955), we have looked more closely at the saponin fraction. We find that there are two saponins, one major and one minor. Both have a branched trisaccharide on C-3 of the sapogenin. Galactose is directly attached to C-3 in both saponins. The major saponin has glucose and arabinose attached to galactose, the minor saponin has glucose and xylose. We suggest the names narthecin and xylosin for the spirostanol form of these two saponins. In fresh juice from leaves we find little narthecin, however. Most of the saponin is present in the furostanol form, with glucose on C-26. Enzymatic hydrolysis showed this glucose to be bound as a β-glucoside. From specific rotations in partial hydrolysates we conclude that the saccharide on C-3 is a β-D-glucoside, α-L-araboside, β-D-galactoside.     

Development of the photosynthetic apparatus during light-dependent greening of a mutant of Chlorella fusca

The formation of chlorophyll, cytochrome f, P-700, ribulose bisphosphate carboxylase as well as photosynthesis and Hill reaction activities were tested during the light-dependent greening process of the Chlorella fusca mutant G 10. Neither chlorophyll nor protochlorophyllide was detected in the darkgrown cells. When transferred to light the mutant cells developed chlorophyll and established its photosynthetic capacity after a short lag phase. In the in vivo absorption spectra a spectral shift of the red absorption peak position from 674 to 680 nm was indicated during the first 3 h of greening. Cytochrome f was already present in the dark-grown cells, but during the greening phase a threefold increase in the cytochrome f content could be seen. At the early stages of greening a characteristic primary oscillation in the content of cytochrome f was observed. P-700 was lacking in the dark and during the first 30 min of illumination. From the first to the second h of light a forced synthesis of P-700 took place and the time-course curve for the ratios of P-700/chlorophyll rose to a sharp maximum. The synthesis of P-700 started together with photosystem I activity and showed similar kinetics. We found the simultaneous appearance of photosystem II, photosystem I, and photosynthetic activities 30 min after the beginning of the illumination. Based on chlorophyll content they attained maximum activity after 2 h of light, but at this time photosystem I capacity proved to be remarkably higher than photosynthetic and photosystem II activities. Highest carboxylase activity existed in darkgrown cells. During the greening process the activity of the enzyme decreased continuously. After 2 h of illumination chlorophyll synthesis partially served to increase the size of the photosynthetic unit, which consequently led to a decrease in the light energy needed to saturate photosynthesis and also to a decrease of photosynthetic rate based on chlorophyll content.Abbreviations Chl chlorophyll - Cyt f cytochrome f - DPIP 2,6-dichlorophenolindophenol - EDTA ethylenediaminetetraacetic acid - GSH glutathione - LH light-harvesting - PS photosystem - RuBP ribulose bisphosphate     

A review of assessment and remediation strategies for hat spot sediments

Sediments at the extreme end of the scale of contamination, designated as hot spot sediments, are considered. The characterization of such hot spots, and an approach for the quantitative assessments of the behaviour and fate of pollutants in such sediments are covered. Experiments with sediments containing extreme levels of heavy metals showed release rates of 56 mg m^–2 d^–1 of dissolved zinc and 0.004 mg m^–2 d^–1 of dissolved mercury. When these sediments were resuspended, the dissolved fluxes were increased by a factor of 2.2 and 128 for zinc and mercury, respectively. The biological implications of hot spot sediments are dealt with, since sediments are an important habitat for many organisms. Various alternatives for clean-up operations including dredging and capping are discussed.