Contribution of sucrose synthase, ADP-glucose pyrophosphorylase and starch synthase to starch synthesis in developing pea seeds

Using genetic variability existing amongst nine pea genotypes (Pisum sativum L.), the biochemical basis of sink strength in developing pea seeds was investigated. Sink strength was considered to be reflected by the rate of starch synthesis (RSS) in the embryo, and sink activity in the seed was reflected by the relative rate of starch synthesis (RRSS). These rates were compared to the activities of three enzymes of the starch biosynthetic pathway [sucrose synthase (Sus), ADP-glucose pyrophosphorylase and starch synthase] at three developmental stages during seed filling (25, 50 and 75% of the dry seed weight). Complete sets of data collected during seed filling for the nine genotypes showed that, for all enzyme activities (expressed on a protein basis), only Sus in the embryo and seed coat was linearly and significantly correlated to RRSS. The contribution of the three enzyme activities to the variability in RSS and RRSS was evaluated by multiple regression analysis for the first two developmental stages. Only Sus activity in the embryo could explain, at least in part, the significant variability observed for both the RSS and the RRSS at each developmental stage. We conclude that Sus activity is a reliable marker of sink activity in developing pea seeds.     

Sea urchin Hox genes: insights into the ancestral Hox cluster

We describe the Hox cluster in the radially symmetric sea urchin and compare our findings to what is known from clusters in bilaterally symmetric animals. Several Hox genes from the direct-developing sea urchin Heliocidaris erythrogramma are described. CHEF gel analysis shows that the Hox genes are clustered on a < or = 300 kilobase (kb) fragment of DNA, and only a single cluster is present, as in lower chordates and other nonvertebrate metazoans. Phylogenetic analyses of sea urchin, amphioxus, Drosophila, and selected vertebrate Hox genes confirm that the H. erythrogramma genes, and others previously cloned from other sea urchins, belong to anterior, central, and posterior groups. Despite their radial body plan and lack of cephalization, echinoderms retain at least one of the anterior group Hox genes, an orthologue of Hox3. The structure of the echinoderm Hox cluster suggests that the ancestral deuterostome had a Hox cluster more similar to the current chordate cluster than was expected Sea urchins have at least three Abd-B type genes, suggesting that Abd-B expansion began before the radiation of deuterostomes.      

Mode of Action of the Sesquiterpene Lactones Psilostachyin and Psilostachyin C on Trypanosoma cruzi

Trypanosoma cruzi is the causative agent of Chagas’ disease, which is a major endemic disease in Latin America and is recognized by the WHO as one of the 17 neglected tropical diseases in the world. Psilostachyin and psilostachyin C, two sesquiterpene lactones isolated from Ambrosia spp., have been demonstrated to have trypanocidal activity. Considering both the potential therapeutic targets present in the parasite, and the several mechanisms of action proposed for sesquiterpene lactones, the aim of this work was to characterize the mode of action of psilostachyin and psilostachyin C on Trypanosoma cruzi and to identify the possible targets for these molecules. Psilostachyin and psilostachyin C were isolated from Ambrosia tenuifolia and Ambrosia scabra, respectively. Interaction of sesquiterpene lactones with hemin, the induction of oxidative stress, the inhibition of cruzipain and trypanothione reductase and their ability to inhibit sterol biosynthesis were evaluated. The induction of cell death by apoptosis was also evaluated by analyzing phosphatidylserine exposure detected using annexin-V/propidium iodide, decreased mitochondrial membrane potential, assessed with Rhodamine 123 and nuclear DNA fragmentation evaluated by the TUNEL assay. Both STLs were capable of interacting with hemin. Psilostachyin increased about 5 times the generation of reactive oxygen species in Trypanosoma cruzi after a 4h treatment, unlike psilostachyin C which induced an increase in reactive oxygen species levels of only 1.5 times. Only psilostachyin C was able to inhibit the biosynthesis of ergosterol, causing an accumulation of squalene. Both sesquiterpene lactones induced parasite death by apoptosis. Upon evaluating the combination of both compounds, and additive trypanocidal effect was observed. Despite their structural similarity, both sesquiterpene lactones exerted their anti-T. cruzi activity through interaction with different targets. Psilostachyin accomplished its antiparasitic effect by interacting with hemin, while psilostachyin C interfered with sterol synthesis.     

MEMBRANE-BOUND NEURAMINIDASE IN THE BRAIN OF DIFFERENT ANIMALS: BEHAVIOUR OF THE ENZYME ON ENDOGENOUS SIALO DERIVATIVES AND RATIONALE FOR ITS ASSAY

—The activity of brain membrane-bound neuraminidase on endogenous and exogenous substrates was comparatively studied in various animals (rat, chicken, rabbit, pig, calf and human). The maximum rate of hydrolysis of endogenous substrates by membrane-bound neuraminidase (using a crude preparation of the enzyme) was different in the various animals (from 0·05 to 0·73 units, referred to 1 mg protein) and was obtained under similar but not identical optimum conditions (pH from 4·1 to 5·1; requirement or not of Triton X-100). The maximum degree of hydrolysis of endogenous substates was also different (from 15 to 27 nmol released NeuNAc/mg protein) and was obtained within different incubation periods (from 2 to 18 h). It corresponded (in rabbit, calf, human brain only), or not, to the actual exhaustion of the endogenous substrates. The endogenous substrates were recognized as both gangliosides and sialoglycoproteins. The extent of hydrolysis of sialoglycoproteins varied from 1·5% in rabbit to 15·6% in chicken brain; the hydrolysis of gangliosides (ranging from 14·1% in pig to 53·7% in rabbit brain) reached only in some animals (rabbit, calf, human) the complete transformation of major oligosialogangliosides into the neuraminidase resistant monosialoganglioside GMI. Upon addition of exogenous substrates (sialyl-lactose, ganglioside GD1a, brain sialopeptides, ovine submaxillary mucin) the actual rate of liberation of total NeuNAc (from both endogenous and exogenous substrates) considerably exceeded, although at a different extent (depending on the animal and on the added substrate used) the rate of hydrolysis of sole endogenous substrates. The possibility of an accurate assay of brain membrane-bound neuraminidase in a crude enzyme preparation is evaluated and guidelines for the assay procedure suggested.     

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.