Myelin basic protein immunohistochemistry: a study of the early stages of myelination in the brainstem of the rat.

An immunohistochemical study of MBP distribution in the brainstem of neonate till 16 d old rats based on the peroxidase-antiperoxidase method is described. Axons already invested with immunoreactive sheaths were found in neonate rats in the ventral funiculus of the cervical spinal cord and in the medial longitudinal fascicle of the medulla oblongata. Fibres commencing with myelination showed a closely spaced array of varicosities in longitudinal sections which diminished gradually. A caudo-rostral decrease in density of myelinated fibres in the brainstem was found in the medial and dorsal longitudinal fascicles. In contrast to other pathways, myelination in the fibres of the corticospinal tract in the brainstem occurred in a strictly synchronized pattern. The same temporal pattern of myelination was also observed in the cervical corticospinal tract, except that a few myelinated fibres had been visible much earlier within the area of the tract. At the exit of cranial nerves, the transitorial zone from central to peripheral myelin was outlined by a decrease in immunostaining.

Comparison of brain ribonucleases of rabbit, guinea pig, rat, mouse and gerbil.

The brain ribonucleases of rabbit, guinea pig, rat, mouse and gerbil were investigated by histochemical and biochemical methods. For the localization, the ribonucleases were electrophoretically transferred from cryostat sections to polyacrylamide gels. Elevated ribonuclease activities were found in the cortex, the basal ganglia, the hippocampal formation and the ventricles, whereas the corpus callosum and the internal capsule exhibited lower activities. The total RNA degrading activities of the brain extracts of the different species varied in a wide range. However, a pre-requisite for the measurement of acid soluble degradation products in the test system was the inactivation of endogeneous ribonuclease inhibitors, present in all extracts. Molecular weight analysis by means of SDS-polyacrylamide gel electrophoresis revealed a characteristic set of ribonucleases for each species, consisting of enzymes with different pH-optima.

The competitive antigen spot test (CAST). A rapid and simple means of quantitatively screening antisera.

A method is described which permits the simultaneous testing of several antisera and an estimation of their antigen binding capacities. The sera to be tested are preabsorbed with different quantities of antigen and subsequently assayed for unbound antibodies by a second incubation with antigen immobilized on nitrocellulose. When the absorbing antigen exceeds a definite concentration, which is specific for a particular antiserum, a clear cut-off is observed when immunostaining is used in the following step. This competitive antigen spot test (CAST) is both quick and easy to perform and was found useful when comparing different antisera.

Occurrence of "stress"-proteins in yeast after heat-shock, acrylonitrile treatment and during the stationary growth phase.

The response of yeast cells to different kinds of "stress" is not identical. Cells of the stationary growth phase synthesize three new proteins of molecular weights 68, 27 and 24 kD, compared with cells of the exponential growth phase, while heat-shocked cells exhibit new proteins of 100, 90, 84, 70 and 24 kD. After treatment with acrylonitrile two new proteins with molecular weights of 70 and 46 kD appear. However, all three kinds of "stress" lead to the induction of a ribonuclease.

Inhibition of ribonuclease activity during RNA synthesis in isolated yeast nuclei by cadmium.

We have developed an efficient transcription system in isolated yeast nuclei. If MnCl2 is substituted by CdCl2, degradation of newly synthesized RNA is markedly reduced. This effect is due to the inhibition of nuclear ribonuclease activity, since microsomal ribonuclease activity is less affected by the cation. The extent to which the addition of CdCl2 to the in vitro transcription assay inhibits ribonuclease activity is demonstrated by the measurements of the size of newly synthesized RNA. Efficient RNA synthesis in this system is not affected up to a concentration of 0.1 M CdCl2.

The occurrence of two ribosomal ribonucleases depending on growth phase in yeast. Induction of ribonuclease in glucose-starved cells.

There are two latent ribonucleases associated with the 40 S subunits of yeast ribosomes which differ in their digestion products, pH optimum, molecular weight, and in their activity during growth phase. The 3'-nucleotide-producing enzyme is active only in the late logarithmic or stationary growth phase, whereas the ribonuclease which produces 5'-nucleotides is present at all growth phases. The enzymes were separated by affinity chromatography and were partially characterized. By changing growth conditions--i.e. decreasing and increasing the glucose concentration in the medium--the activity of the 3'-ribonuclease could be induced or reduced.

The induction of a ribosomal ribonuclease in Saccharomyces cerevisiae.

The ribosomal ribonuclease of yeast is induced after a withdrawal of glucose. Cycloheximide inhibits the synthesis of the enzyme under conditions of induction but antimycin shows no effect. Hence, the increase of the ribonuclease activity during growth of yeast is due to newly synthesized enzyme, and the induction does not depend on respiration.

A ribonuclease from yeast associated with the 40 S ribosomal subunit.

1. Autodegradation of yeast ribosomes is due to a 'latent' ribonuclease which is associated with the 40 S ribosomal subunit. 2. The ribonuclease was extracted in the presence of EDTA from ribosomes and purified 118-rold by protamine sulphate precipitation, (NH4)2SO4 fractionation and chromatography on DEAE-cellulose. 3. The optimum pH for this enzyme is 5 to 6.5 while the optimum temperature is 45 to 50 degrees C. Incubation for 10 min at 60 degrees C caused a reduction in enzyme activity of 70%. 4. The ribonuclease has an endonucleolytic activity against rRNA, tRNA, poly(A), poly(U) and poly(C) but does not degrade poly(G) or DNA. It hydrolyzes the homopolymers to nucleoside 3'-phosphates. 5. Zn2+, Mn2+, heparin, glutathione and p-chloromercuribenzoate inhibit the ribonuclease, while Na+, K+, EDTA and sermidine have only little or no effect. 6. It binds tightly to yeast ribosomes but only loosely to ribonuclease-free wheat germ ribosomes. 7. Polyribosomes possess less autodegradation activity than monoribosomes, isolated from the same homogenate.

Photodynamic action of thiopyronine on polyribosomes and cell-free protein synthesis in yeast.

The treatment of yeast with thiopyronine (TP) caused a degradation of polyribosomes, even if the cells were not illuminated. In contrast, no differences in the polyribosome profiles of illuminated and unilluminated cells could be seen. Likewise, in in vitro experiments, there was no degradation of polyribosomes caused by dark effect or photodynamic action. Cell-free protein synthesis was inhibited up to 75 per cent by the photodynamic effect when the complete system was treated, but only up to 40 per cent when the polyribosomes or enzyme fraction were treated. Since the enzyme fraction contained aminoacyltRNA-synthetases and tRNA, it was necessary to investigate separately the effect of TP on the enzyme and the tTNA. It was shown that the aminoacyl-tRNA-synthetase and not tRNA was effected by the photodynamic action in its biological activity.

[Initiation in a polyribosome-dependent protein-synthesizing cell-free system from Saccharomyces (author's transl)]. / Initiation in einem Polyribosomen-abhängigen Protein-synthetisierenden zellfreien System aus Saccharomyces

A method to prepare polyribosomes from yeasts by using the french-press is described. The highest yield of polyribosomes was derived from late log-phase cells. These polyribosomes, incubated in a cell-free system, were able to reinitiate protein synthesis, which was shown by inhibiting aminoacid incorporation by aurintricarboxylic acid, edeine and sodiumfluoride. We developed the translational system in order to look for the optimal ion-conditions of a DNA-dependent protein-synthesizing system. We found out that at the optimal MgCl2-concentration (6 mM) protein synthesis was strongly inhibited by Mangan ions which are required for transcription in yeast. If protein-synthesis was carried out with 2 mM and 3 mM MgCl2 maximal aminoacid incorporation was observed at 2 mM and 1.5 mM MnCl2.