On the membrane translocation of diphtheria toxin: at low pH the toxin induces ion channels on cells.

Diphtheria toxin (DT) in acidic media forms ion-conducting channels across the plasma membrane and inhibits protein synthesis of both highly and poorly DT-sensitive cell lines. This results in loss of cell potassium and in entry of both sodium and protons with a concomitant rapid lowering of membrane potential. The pH dependency of the permeability changes is similar to that of the inhibition of cell protein synthesis. DT-induced ion channels close when the pH of the external medium is returned to neutrality and cells recover their normal monovalent cation content. Similar permeability changes were induced by two DT mutants defective either in enzymatic activity or in cell binding, but not with a mutant defective in membrane translocation. The implication of these findings for the mechanism of DT membrane translocation is discussed.     

Distribution of Thiamine, Thiamine Phosphates, and Thiamine Metabolizing Enzymes in Neuronal and Glial Cell Enriched Fractions of Rat Brain

The distribution of thiamine, thiamine phosphoesters, and the thiamine pyrophosphate synthetizing [thiamine-pyrophosphokinase (TPKase)] as well as hydrolyzing [thiamine pyrophosphatase (TPPase) and thiamine monophosphatase (TMPase)] enzymes was determined in neuronal and glial enriched fractions prepared from rat brain. Nucleoside diphosphatases [inosine diphosphatase (IDPase) and uridine diphosphatase (UDPase)] and nucleoside monophosphatases [uridine monophosphatase (UMPase) and inosine monophosphatase (IMPase)] were also determined. Thiamine and thiamine mono- and pyrophosphate were present in neuronal enriched fractions at concentrations 2.8, 3.6, and 4.6 times higher than in glial fractions. TMPase was found only in glial enriched fractions, whereas the levels of TPKase, UMPase, IMPase, IDPase, UDPase, and TPPase were 2.0-, 2.2-, 1.3-, 2.8-, 3.7-, and 20.8-fold higher in neuronal than in glial fractions.     

Transient Photoresponses of a Phototactic Microorganism, Haematococcus Pluvialis, Revealed by Light Scattering

A new method, using incoherent light scattering, has been developed to measure the flagellar beating frequency of swimming microorganisms. By means of this method, transient changes of flagellar beating frequency in response to white light flashes have been revealed in samples of a phototactic microorganism, Haematococcus pluvialis. An increase of flagellar beating frequency occurs when the flash dose (flash intensity × flash duration) is sufficient. Reciprocity between light intensity and flash duration holds for durations not exceeding 60-80 ms. For lower doses a bimodal distribution of flagellar beating frequency is revealed. No effect is observed for very low flashes or for red stimuli, whereas green light is effective. A detailed analysis of experimental results has allowed us to determine the characteristic time of the effect and follow its evolution. The correlation of this effect with visually observed behavior is discussed and a possible underlying mechanism is suggested.     

Hydrophobic photolabelling of sarcoplasmic reticulum with [125I]TID

[125I]TID, a small photoreactive lipophylic reagent, was used to label intrinsic proteins of rabbit and rat sarcoplasmic reticulum membranes. A 160,000 glycoprotein, the Ca2+-ATPase and polypeptides of mol. wt 53-55,000, 30,000, 20,000 and 6000 dalton were labelled suggesting that these proteins are integral membrane components.     

Flow cytometric evaluation of proliferative activity and ploidy in myelodysplastic syndromes and acute leukemias

Propidium iodide (PI) DNA distribution of bone marrow (BM) cells was studied by flow cytometry (FCM) in 36 patients without hematologic or malignant disease (normal BM) and in 172 patients with anemias (36 pts), myelodysplastic syndromes (MDS) (33 pts) and acute leukemia (AL) at diagnosis (60 pts), remission (24 pts) and relapse (19 pts). White blood cells from normal male subjects were used as an external diploid reference standard (median CV = 3.8). Patients with normal BM, anemias, MDS and acute leukemia at diagnosis had tritiated thymidine labeling index (LI) and most with MDS and AL had also evaluable cytogenetics performed on the same BM sample used for FCM. In normal BM, median aliquot of cells with PI-DNA content intermediate between the diploid and the tetraploid value (2n-4n cells %) was 15.7. The ratio between the fluorescence intensity of the G0/1 peak of normal BM cells and the fluorescence intensity of the G0/1 peak of the reference standard (FI ratio) ranged from 93 to 1.05 (mean +/- 2SD). The 2n-4n cell % was higher than normal in anemias (p less than .001), lower in leukemias (p less than .001) and widely scattered in MDS. A linear correlation was found between 2n-4n cell % and LI, with 2n-4n cell % value higher than LI. The FI ratio was lower than normal in anemias (p less than .05), higher in AL with normal cytogenetics (p less than .02) and broadly scattered in MDS with normal cytogenetics. From our experience, PI-DNA-FCM is a simple and adequate method to evaluate proliferative activity in hematologic diseases. Nevertheless, caution must be taken in attributing small changes in FI ratio to aneuploidy, since they are found in anemias and in MDS and AL with normal cytogenetics, possibly due to differences in PI uptake by different cell types.     

Tubular structures associated with the endothelial endoplasmic reticulum in glomerular capillaries of Rhesus monkey and nephritic man

Summary Round bodies, tubular profiles and crystalline images have been studied by electron microscopy in the endothelium of seven normal young Rhesus monkeys and in the renal glomerular endothelium of two nephritic human patients. The crystalline images are most frequently formed by aggregation of round bodies, 200–240 Å in diameter. In Rhesus monkeys a variety of crystalline images are seen. On the contrary, in nephritic patients round bodies and tubular profiles are rare and less organized. In the glomerular endothelium of two normal men they were not found.The results obtained suggest that the round bodies, the tubular profiles and the crystalline images result from sectioning of a system of undulating tubules associated with the smooth endoplasmic reticulum. In nephritic patients the formation of such a tubular system could represent a change taking place within the affected cells as a pathologic response to the disease.This work was supported by U.S. Public Health Service (N.H.I.), Grant AI-04527-03-04, and by the Consiglio Nazionale delle Ricerche (C.N.R.), Contributo 115/0815/0-1365. The Authors are greatly indebted to Miss Hermina Spiele for skilful technical assistance.     

A re-evaluation of the mesangial cells of the renal glomerulus

Summary The topographical localization of the mesangial cells in renal glomeruli of rats, and their relationships with the structures at the hilus of the glomerulus were studied in this investigation. It was observed that the mesangial cells occupy a parietal position in the wall of the glomerular capillaries, and that they are anatomically continuous with the smooth muscle cells of the tunica media of the glomerular arterioles.This study was supported by a United States Public Health Service Grant AM 08628 (Institute of Arthritis and Metabolic Diseases).     

Stomatal and cuticular transpiration of beans (Phaseolus vulgaris M.) attacked by rust (Uromyces appendiculatus [Pers.] Link)

The changes of stomatal and cuticular transpiration of bean plants were investigated by graphical transpiration curves method (Slavík 1958). Bean leaves were infected by fungusUromyces appendiculatus (Pers.) Link. After the infection the intensity of stomatal transpiration had a decreasing tendency. Beginning with the sixth day after infection, the proportion of stomatal and cuticular transpirations becomes more expressive, i.e. the leaves transpire more by cuticles than by stomata. The higher share of cuticular transpiration brings extensive water relations to the diseased plants.     

Histidine 21 is at the NAD+ binding site of diphtheria toxin

Treatment of fragment A chain of diphtheria toxin (DT-A) with diethylpyrocarbonate modifies His-21, the single histidine residue present in the chain, without alteration of other residues. Parallel to histidine modification, NAD+ binding and the NAD-glycohydrolase and ADP-ribosyltransferase activities of DT-A are lost. Both NAD+ and adenosine are very effective in protecting DT-A from histidine modification and in preserving its biological properties, while adenine is ineffective. Reversal of histidine modification with hydroxylamine restores both NAD+ binding and enzymatic activities of the toxin. The possible role of His-21 in the activity of diphtheria toxin is discussed in relation to the available three-dimensional structure of the related toxin produced by Pseudomonas aeruginosa.