Mycoplasma development and cell alterations in white clover affected by clover dwarf an electron microscopy study

Summary The presence of mycoplasma has been demonstrated in the phloem of leaves of white clover (Trifolium repens L.) affected by clover dwarf. Mycoplasma-like bodies were found both in parenchyma and companion cells and in sieve elements.In young parenchyma and companion cells mycoplasma-like bodies appeared as round or oval particles with high ribosomal content, delimited by a ribosome-bearing membrane. Their diameter ranged between 50 and 400 nm. In mature sieve elements they were larger, more pleomorphic, and showed a central clear area containing presumed DNA filaments. Budding and dividing forms were sometimes seen among them.The main alterations found in the infected cells were: increased ribosome content, dilation of the perinuclear space, degeneration of mitochondria and chloroplasts, and cytoplasmic vacuolation. Many cells appeared completely disrupted and their content was replaced by a great number of pleomorphic mycoplasma.This investigation was supported by a grant of Consiglio Nazionale delle Ricerche, Rome.     

Rate-determining step in phospholipase A2 mechanism. 18O isotope exchange determined by 13C NMR

H2(18)O isotope exchange into specifically 13C-labeled substrate was used to obtain information on the rate-limiting step in the action of the phospholipase A2 from the venom of the Indian cobra (Naja naja naja). Incorporation of 18O was detected by the effect of 18O on 13C chemical shifts in 13C NMR. The enzymatic hydrolysis of a micellar phosphatidylcholine analogue of platelet-activating factor 1-alkyl-2-[1-13C]lauroyl-sn-glycero-3-phosphorylcholine proceeds by an O-acyl cleavage of the sn-2 ester bond. The reaction was examined for simultaneous 18O incorporation into the substrate. No exchange was found, suggesting that the hydrolytic step is not followed by a higher energy transition state and that it or a step before it appears to be rate-limiting. Previous experiments on phosphatidylethanolamine activation indicate that kcat is altered but that the km remains the same upon activation, suggesting that the binding steps occurring before the hydrolytic step are not affected. This strongly suggests that the hydrolytic step is in fact the rate-limiting step under these conditions. The 13C, 18O NMR technique should be generally applicable to mechanistic questions of this type.     

Immunocytochemical localization of pancreatic carboxylic ester hydrolase in human paneth cells

Summary The protein-A gold method using specific rabbit sera directed against pure human pancreatic chymotrypsinogen and carboxylic ester hydrolase was applied to locate these (pro)enzymes in human pancreatic acinar cells and intestinal Paneth cells. Quantitative evaluation of the labelling indicated that both (pro)enzymes are present in pancreatic acinar secretory granules. In Paneth cell secretory granules, only carboxylic ester hydrolase was present in significant amounts, although the labelling for this enzyme was less intense than that observed in pancreatic zymogen granules. The results obtained support the view that Paneth cells represent a diffuse exocrine gland scattered along the intestine, whose role is either to act as a substitute in the event of a deficient pancreas or to regulate the intestinal flora.     

Effect of sucrose diet on insulin secretion in vivo and in vitro and on triglyceride storage and mobilisation of the heart of rats

Basal heart triacylglycerol (TG) (mumole triacylglycerol/g of dry weight) (- before "in vitro" Langendorff perfusion -) was significantly higher in animals rendered chronically hypertriglyceridaemic (H) by a 63% sucrose-rich diet than in controls (C, standard diet); 28 +/- 2.6 means + SEM vs. 19.3 +/- 1.2; respectively (p less than 0.01). After 40' perfusion with Krebs-Henseleit buffer + 5.5 mM glucose, 2.5 mM Ca++, TG content fell to 14.2 +/- 0.6 in C and 14.9 +/- 1.9 in H (n.S.). Administration of 1 n mol x min-1 of glucagon (Gn) from min 20 to 40 reduced TG to 9.0 +/- 0.5 in C (p less than 0.05). In contrast no effect of Gn was observed in H (TG at min 40: 16.7 +/- 2.5). Glycogen (Gly) content (mumol/g of dry weight) after Gn perfusion fell from 30 +/- 1.9 to 17 +/- 2.1 (p less than 0.01) in C, while again no effect was recorded in H. "In vivo" plasma glucose fractional coefficient disappearance rate was lower (p less than 0.001) in H: 1.01 x 10(-2) +/- 0.09 x 10(-2) vs 2.61 x 10(-2) +/- 0.14 x 10(-2) in C, in spite of H showing hyperinsulin secretion. Hyperinsulinism was further documented by "in vitro" Iri release studies from incubated pancreas pieces. In the absence of glucose (G) from the incubation medium H produced 541 +/- 19.8 mU/mg weight Tissue/20', while C produced 91.2 +/- 12.7 (p less than 0.001). With 100 mg% G, H released 1058 +/- 259 and C 377 +/- 82.5 (p less than 0.001). It is suggested that hyperinsulin secretion plus insulin resistance may account for the above findings.     

Use of a particular cellulose acetate supporting agent in the electrophoretic evaluation of human hemoglobin

576 subjects of whom 450 with hereditary anaemia and 116 normal are studied to establish the haemoglobin pattern. The assay is carried out using the standard cellulose acetate and an particular cellulose acetate medium cellogel RS "Wedge". The results show that cellogel RS in comporation with standard medium permits either an better resolution of the hemoglobin bands or a better detection of the pathologic bands.     

Bidimensional electrophoresis of factor VIII antigen on cellulose acetate

It is described two-dimensional immunoelectrophoresis using cellulose acetate as supporting medium compared with agarose gel method. The results show that the determination of the FVIIIR: Ag on cellulose acetate is a technique more simple and rapid than agarose gel method and many free from the technical failure.     

Histological localization of citrus infectious variegation virus (CVV) inPhaseolus vulgaris

Summary Ultrathin sections ofPhaseolus vulgaris leaves were studied in the electron microscope. The leaves were taken from plants, both healthy and experimentally infected with CVV. The sieve tubes and companion cells of all samples contained a slime-like substance, more or less organized into compact systems. In the mature sieve elements of virus-infected plants systems of parallel membranes were seen along which spherical particles, of about 30 m diameter, were aligned in simple rows.Pubbl No. 117 of Gruppo di Ricerca per le Virosi, del C. N. R.     

A procedure for selecting mammalian cells with an impairment in oxidative phosphorylation.

The mitochondrial encephalomyopathies in man are characterized by heterogeneous defects leading to an impairment in the pathway of aerobic energy production. As a means of investigating the molecular and genetic mechanisms underlying these disorders we have developed a procedure for selecting mammalian cell lines with features resembling the human pathological phenotypes. The principle of the selection is the use of a fluorescent amphiphilic dye, 2,4-(dimethylamino)-1-styrylmethylpyridiniumiodine, a cation showing two main features. Firstly, it is accumulated by mitochondria to an extent correlated with the magnitude of the electrochemical gradient of protons across the mitochondrial inner membrane. Secondly, upon irradiation with UV light, it gives rise to formation of free radicals, which inflict damage to the cell. Mutant cells with an impairment in oxidative phosphorylation will have more chance to survive than wild type cells. The selection procedure was applied to a stock of mutagenized Chinese hamster ovary cells. After subcloning of the cells which survived the selection procedure, twenty-six independent clones were isolated. Eighteen of the clones had a partial deficiency of cytochrome c oxidase ranging from 30 to 60% of the activity in control cells. The properties of two of the clones are described. One clone has been cultured under non-selective conditions for at least 12 months with retention of the partial deficiency of cytochrome c oxidase.