Structural and Functional Impact of SRP54 Mutations Causing Severe Congenital Neutropenia

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Regulation of the photosynthetic electron transport during dark-light transitions by activation of the ferredoxin-NADP+-oxidoreductase in higher plants

Absorbance changes associated with the oxidation and reduction of cytochrome f belong to the classical observations about the interaction of the two photosystems. A complex induction pattern of cytochrome f oxidation results, if both photosystems are excited simultaneously. This indicates a light-modulated regulation of the photosynthetic electron transport, which we examined for intact biological systems of decreasing complexity. The ferredoxin-NADP⁺-oxidoreductase (FNR) is suggested to be activated by light and inactivated in the dark. This is pointed out by the kinetics of variable fluorescence and by the influence of different artificial electron acceptors on the cytochrome f kinetics. The photoreduction of NADP⁺ by carefully prepared thylakoids demonstrates the activation process directly.This work was supported by the Deutsche Forschungsgemeinschaft.     

12-O-tetradecanoyl phorbol 13-acetate stimulates the myristylation of an approximately 82 kDa protein in HL-60 cells

We have studied protein acylation using [3H]myristate in the two leukemia cell lines HL-60 and HL-60 Blast II. The latter is a variant which does not differentiate after treatment with 12-O-tetradecanoyl phorbol 13-acetate (TPA). The acylation profiles of the two cell lines as examined by SDS-PAGE differed. TPA induced the myristylation of an approximately 82 kDa protein in the sensitive cells, but not in the resistant cells. Myristic acid was shown to be covalently linked to these proteins. Analysis of the cell lipids labelled with [3H]myristate showed that in contrast to observations with the proteins, the changes induced by TPA were observed in both TPA-sensitive and TPA-resistant cells. We conclude that the induction of myristylation may be an important step in the mechanism of differentiation.     

Parathyroid cell variants may be provoked during immersion fixation

Summary Parathyroid glands of cattle, dogs, cats, mice and rats were immersed in glutaraldehyde or mixtures consisting of glutaraldehyde, formaldehyde and acrolein in either Na-phosphate, Na/K-phosphate or Na-cacodylate buffer, and postfixed with OsO₄ in the same buffers or, alternatively, in s-collidine.Excellent preservation of bovine, feline and murine parathyroid glands was achieved with fixation mixtures containing 1% glutaraldehyde, 1.5–2% formaldehyde and 2.5–5% acrolein in 0.1 M Na-cacodylate with or without Ca²⁺ and Mg²⁺, Na-phosphate or Na/K-phosphate at 4°C followed by postfixation with 1% OsO₄ in the same buffers or in s-collidine containing sucrose, Ca²⁺ and Mg²⁺. This procedure largely abolished the occurence of parathyroid cell variants. Bovine parathyroid glands were also satisfactorily preserved with 1% glutaraldehyde and 2% formaldehyde whereas 1% glutaraldehyde and 2.5 or 5% acrolein, lower or higher buffer osmolarity, or immersion at room temperature led to vacuolization of RER and to breakdown of membranes. In contrast, all fixation protocols led to the formation of dark and light cell variants and to multinucleated syncytial cells in dog and rat parathyroids. The results thus show that parathyroid cell variants arise during immersion fixation and that aldehydes, buffers and temperature are important factors for provoking parathyroid cell variants.     

Potency of microwave irradiation during fixation for electron microscopy

Liver, skeletal muscle, peripheral nerves, pancreas, thyroid and adrenal cortex were prepared for electron microscopy employing microwave energy either during prefixation with glutaraldehyde or instead of prefixation. Microwave irradiation in the presence of glutaraldehyde in Na/K-phosphate or Na-cacodylate containing CaCl2 and MgCl2 led to distinct appearance of membranes, mainly plasma membrane, and membranes of SER, Golgi complex and mitochondria in liver, pancreas and muscle. The area of high quality fixation, however, was limited to the periphery of samples. On the other hand, SER was dilated in cells of the adrenal cortex, and RER markedly vacuolated in thyroid follicular cells. Microwave irradiation in the presence of Na/K-phosphate and subsequent osmication resulted in preservation of the ultrastructure in similar quality as was obtained by osmication without previous immersion in glutaraldehyde. However, the preservation of SER and Golgi complex in liver and pancreas, and of mitochondria in muscle was greatly improved. Small myelin sheaths remained intact whereas large ones showed focal disintegration. We consider that enhancement of fixation by microwave energy may greatly improve preservation of membranes in some tissues. Successful fixation depends on the use of glutaraldehyde during microwave irradiation, the type of buffer, the addition of ions to increase stabilization, the exposure time to heat, and on postosmication.     

The Loss of Structural Integrity in Damaged Spruce Needles from Locations Exposed to Air Pollution I. Mesophyll and Central Cylinder

Abstract In connection with the new type of forest damage, the individual disease situation of two-year-old spruce ( Picea abies ) needles was analyzed histopathologically in forest areas exposed to different levels of O₃-, SO₂- and NO₃- pollution.
Early damage results from losses of chlorophyll in the mesophyll cells. The bleaching is more intensive towards the apex in severely damaged needles. The cytoplasm is aggregated at the cell wall and the chloroplasts show definite structural damage as well.
The mesophyll cells below the epidermis, or the cells adjacent to the vascular bundle sheath, appear to be particularly susceptible. Collapsed cells (bone cells), which increase in number with damage, can lead to tissue death in certain needle areas, (brown tips, transverse bands).
Necrotic spots are manifested as groups of dissociated cells in which hypertrophic and collapsed cells as well as abnormal proliferations can be observed.
Hypertrophy and cell collapse appear in the central cylinder in addition to severe phenol deposits.
Bone cells and chlorophyll losses can already be detected in the green needles of damaged trees, indicating latent damage, which becomes macroscopically visible only after more extensive damage.
Our results indicate that no biotic stress factors take part in the damage of the spruce needles investigated here. Anthropogenic air pollutants in addition to abiotic stress factors must be regarded as a main cause of damage.     

Relationship between membrane sterol composition and responsiveness to 12-O-tetradecanoylphorbol 13-acetate in HL-60 human promyelocytic leukaemia cell lines.

We have examined the sterol composition and metabolism of promyelocytic leukaemia cell lines (HL-60) after treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). A variant cell line (Blast II cells) which is resistant to TPA was used as control. Analysis of the sterols of TPA-sensitive cells radiolabelled with [3H]leucine, [14C]acetate or [14C]pyruvate showed a high incorporation into cholesterol and a low incorporation in lanosterol + dihydrolanosterol. The inverse relationship was observed in TPA-resistant cells. Experiments with other cellular variants representing TPA-sensitive and TPA-resistant classes gave similar results. Analysis of the cellular sterol composition by gas chromatography confirmed that TPA-resistant cells are particularly rich in lanosterol/dihydrolanosterol. TPA treatment enhanced the incorporation of [14C]pyruvate into the sterol fraction of both cell types. This was accompanied by an alteration of incorporation into several lipids, particularly phospholipids. Pulse-chase studies with [14C]acetate revealed that TPA induced the release of radioactive lipids into the medium from HL-60 and Blast II cells. However this treatment released phospholipids from the TPA-sensitive cells and sterols and fatty acids from the TPA-resistant cells. We conclude that the sterol composition can regulate specific biochemical processes in the membrane and can be considered as a factor that plays a role in the responsiveness of HL-60 cells to TPA.