F.K. De Graaf

Identification of motoneurons in the spinal cord of the zebrafish (Brachydanio rerio), with special reference to motoneurons that innervate intermediate muscle fibers

SummaryWe investigated the location and size distribution of motoneurons that innervate red, intermediate and white muscle fibers in the axial musculature of the zebrafish (Brachydanio rerio). Motoneurons were identified by retrograde labeling from the respective myotomal compartments with horseradish peroxidase applied either in small polyacrylamide gel fragments or as pure crystals. We found a spatial relationship between the three myotomal muscle fiber types and the corresponding motoneurons. The white motoneurons are grouped in the dorsal part of the motorcolumn, near the central canal. Motoneurons of the red and intermediate muscle are clustered in the ventral part of the motorcolumn. The average position of the red motoneurons is ventral to that of the intermediate motoneurons. Soma sizes are distributed over wide overlapping ranges, spanning from 41 to 352 μm2 for red and intermediate and from 56 to 894 μm2 for white motoneurons. These data are discussed in relation to the recruitment order of myotomal muscle fibers of different types as revealed by electromyographic recordings in fish, and the so called “size principle” for motoneuron recruitment.

Mode of action of a bacteriocin produced byEnterobacter cloacae DF 13

Enterobacter cloacae DF 13 produces a bacteriocin with killing action onKlebsiella edwardsii var.edwardsii. The degree of sensitivity to the bacteriocin depended on the medium in which the cells were grown and on the bacteriocin concentration used. An excess of bacteriocin (60 K.U./ml) arrested growth in about 60 min. Growth of bacteriocin-treated cultures could be restored by trypsin treatment. In Brain Heart Infusion cultures trypsin rapidly restored bacterial growth even after 60 min of bacteriocin treatment. However, in broth cultures and minimal medium cultures treated with bacteriocin for only 10 min, it took 4 to 5 hr before growth started again. The bacteriocin had little effect on resting cells. Broth-grown cells had about 280 and BHI-grown cells about 340 bacteriocin receptor sites. Bacteriocin DF 13 strongly inhibited protein synthesis after a lag-time of 15 to 60 min depending on the concentration used but had no effect on RNA and DNA synthesis nor on respiration and fermentation. The bacteriocin stimulated RNA synthesis in a leucine-deficient mutant after leucine deprivation.

Effect of a bacteriocin produced by Enterobacter cloacae on protein biosynthesis.

Abstract 1. 1. Bacteriocin DF 13 , produced by Enterobacter cloacae strain DF 13 , inhibits protein synthesis in Enterobacter cloacae strain 02, while RNA and DNA synthesis continue at a reduced rate. 2. 2. Ribosomes from bacteriocin-treated cells are unable to support phage MS 2 -RNA directed incorporation of 14 C-labeled amino acids. The ribosomes retain the ability to bind mRNA, but appear not to bind fMet-tRNA fMet under direction of MS 2 -RNA. 3. 3. Ribosomes from bacteriocin-treated cells are physically intact with respect to their sedimentation behaviour in sucrose gradients, the integrity of their RNA components, and the composition of ribosomal proteins. 4. 4. Addition of bacteriocin to exponentially growing cells induces a leakage of K + and an accumulation of 30-S and 50-S ribosomal subunits with a concomitant decrease in larger ribosomal particles.

Mode of action of the cloacin DF13-immunity protein.

1. Cells of Enterobacter cloacae harbouring the bacteriocinogenic factor Clo DF13 produce an immunity protein which inbhbits the in vitro activity of cloacin DF13. The amino acid composition of purified immunity protein was determined. 2. Experiments about the protection of ribosomes against cloacin DF13 in the presence of the immunity protein show that one molecule of immunity protein neutralized the activity of one molecule cloacin. 3. Direct and specific interaction of cloacin DF13 with the immunity protein has been demonstrated by the analysis of mixtures of both proteins on polyacrylamide gels and by changes in the fluorescence response of cloacin DF13-bound 1-anilinonaphthalene-8-sulfonate in the presence of immunity protein.

Effects of cloacin DF13 on the functioning of the cytoplasmic membrane

Adsorption of cloacin DF13 onto sensitive cells induces a leakage of potassium ions after a lag of 15–20 min. The rate of the potassium efflux is proportional to the bacteriocin concentration used. The extent of the potassium loss depends on the concentrations of bacteriocin and of extracellular potassium. Leakage of potassium ions is accompanied by an uptake of sodium ions. Cloacin DF13 does not affect the transport of magnesium ions, phosphate ions or amino acids. About 10 min after addition of the bacteriocin the ATP content of the cells begins to decrease gradually to about 25% of the original level. Anaerobiosis, lack of energy source and inhibition of oxidative phosphorylation may protect the cells if applied within 10 min after addition of the bacteriocin. The results suggest that the action of cloacin DF13 proceeds through at least two distinct phases. Phase I is a period after bacteriocin adsorption which is reversible and in some way depends on oxidative phosphorylation. This phase does not cause any cellular damage. Phase II is irreversible and results in a disturbance of several cellular functions.

Purification of a bacteriocin produced by Enterobacter cloacae DF13

Abstract 1. 1. Bacteriocin Df13 was purified from Enterobacter cloacae Strain DF 13, by (NH 4 ) 2 SO 4 precipitation and ion-exchange chromatography on QAE-Sephadex. 2. 2. Polyacrylamide gel electrophoresis, isoelectric density gradient electrophoresis and amino acid analysis indicated that the bacteriocin preparation was homogeneous. 3. 3. Chemical analysis showed that the bacteriocin is a simple protein composed exclusively of amino acids. 4. 4. The molecular weight of the bacteriocin calculated from the amino acid analysis is 56 000 ± 1000. 5. 5. The isoelectric point of the bacteriocin is 9.75 ± 0.02. 6. 6. The number of bacteriocin molecules per killing particle is about 12.

Histochemical profiles of motoneurons innervating muscle fibres with different activity patterns in the zebrafish, Brachydanio rerio

SummaryEnzyme histochemical profiles of spinal motoneurons in the zebrafish were determined. Five enzymes of glucose metabolism were chosen: glucose-6-phosphate dehydrogenase (G6PDH), hexokinase (HK), phosphofructokinase (PFK), succinate dehydrogenase (SDH) and NADH tetrazolium reductase (NADH-TR). Motoneurons were traced with Fluorogold and classified as those that innervate white muscle fibres (W-MNs) and those that innervate red and intermediate muscle fibres (R/ I-MNs). The average enzyme activities per volume of tissue in the somata of both populations differed at most by 25%. Both the average soma volume and the average number of muscle fibres innervated are three times larger for the W-MNs than for the a/I-MNs. This suggests that the total amount of enzyme activity within a neuron soma matches target size.In the R/I-MNs, the activities of SDH and NADH-TR were closely correlated (correlation coefficient, r=0.99;p<0.05) and HK activity correlated well with G6PDH activity (r=0.94;p<0.05), butnot with PFK (r=0.64;p>0.05). In the W-MNs, there was no correlation between SDH and NADH-TR (r=−0.59;p>0.05) or between HK and G6PDH (r=0.50;p>0.05) and the correlation coefficient between HK and PFK activity was close to zero (r=0.04;p>0.05).It was concluded that in the R/I-MNs gwhich are continuously ctive, firing activity is fuelled by oxidative metabolsm. We suggest that in the W-MNs glucose is stored in the form of glycogen and that, despite high levels of NADH-TR present, the energy for intermittent firing activity is provided by glycolysis.

Enzyme histochemical profiles of fish spinal motoneurons after cordotomy and axotomy of motor nerves

Histochemical profiles were made of identified spinal motoneurons from normal adult zebrafish and from animals subjected to cordotomy or unilateral axotomy of the motor nerves. The lesions caused an increase of the myotomal area with oxidative muscle fibers. We studied the question: do changes in the myotomal muscle configuration concur with changes in the enzyme histochemical profiles of innervating motoneurons? Based on the location and size of cell somata, two categories of motoneurons are distinguished: large white (W) motoneurons that innervate the deep fast, glycolytic muscle fibers, and smaller red and intermediate (RI) motoneurons that innervate the superficial slow oxidative and intermediate muscle fibers. In normal animals, glucose-6-phosphate dehydrogenase activity is high in the large W motoneurons and relatively low in the small RI motoneurons. The reverse holds for succinate dehydrogenase activity is high in the large W motoneurons and relatively low in the small RI motoneurons. The reverse holds for succinate dehydrogenase activity. W and RI motoneurons show similar nicotinamide adenine dinucleotide diaphorase activity. Short- (2 weeks) and long- (8 weeks) term effects of lesions were studied. The results show that: (1) the 3 types of lesions lead to prolonged changes in the enzyme histochemical profiles of spinal motoneurons. The type of change depends on the type of lesion and on the type of motoneuron; (2) unilateral axotomy of the motor nerves affects the histochemical characteristics of spinal motoneurons and the myotomal muscle fiber type configuration on the ipsi- and contralateral side. The contralateral effects are conceived as adaptations to maintain a left-right symmetry in the motor output.

Effects of temperature on the activity of cloacin DF13 and cloacin DF13-immunity protein.

During the interaction of cloacin DF13 and sensitive cells the cloacin molecules display different functions which can be distinguished on the basis of their heat-sensitivity. Binding to cell envelope receptors, binding of immunity protein and in vitro inactivation of ribosomes are heat-stable functions in contrast with the entire killing action in vivo. Cloacin DF13-immunity protein appears to be a heat-stable inhibitor of the fibosome inactivation caused by cloacin DF13.

ULTRASTRUCTURAL CHARACTERISTICS OF ZEBRAFISH SPINAL MOTONEURONS INNERVATING GLYCOLYTIC WHITE, AND OXIDATIVE RED AND INTERMEDIATE MUSCLE FIBERS

Summary Spinal motoneurons in the zebrafish were classified using morphological criteria. Dorsomedial white motoneurons which innervate the fast, glycolytic white muscle fiber compartment were distinguished from ventrolateral red and intermediate motoneurons which innervate the slow, oxidative, red and intermediate muscle fiber compartments. Synapses on cell somata and cell organelles were studied in detail. The motoneurons which innervate white muscle fibers (W motoneurons) are considerably larger than those which innervate red and intermediate muscle fibers (RI motoneurons; W > RI). Significant differences were also found in the size of the nucleus (W > RI) and in the ratio size nucleus/size soma (W RI) and mitochondria (W RI). Terminals with dense cored vesicles appeared on W motoneuron somata only. Within the motoneuron population, there was a positive correlation between the coverage of terminals containing flat vesicles and the perimeter of the cell soma. In RI motoneurons, there was a positive correlation between the perimeter of the cell and the amount of endoplasmic reticulum. A negative correlation was found between the RI cell perimeter and mitochondria, which is in line with a high succinate dehydrogenase activity in small cells.