ShiftDetector: detection of shift mutations

MOTIVATION: Sequencing of a bi-allelic PCR product, which contains an allele with a deletion/insertion mutation results in a superimposed tracefile following the site of this shift mutation. A trace file of this type hampers the use of current computer programs for base calling. ShiftDetector analyses a sequencing trace file in order to discover if it is a superimposed sequence of two molecules that differ in a shift mutation of 1 to 25 bases. The program calculates a probability score for the existence of such a shift and reconstructs the sequence of the original molecule. AVAILABILITY: ShiftDetector is available from http://cowry.agri.huji.ac.il     

Mutations in short stature homeobox containing gene (<Emphasis Type="Italic">SHOX</Emphasis>) in dyschondrosteosis but not in hypochondroplasia

Dyschondrosteosis (DCO) and hypochondroplasia (HCH) are common skeletal dysplasias characterized by disproportionate short stature. The diagnosis of these conditions might be difficult to establish especially in early childhood. Point mutations and deletions of the short stature homeobox containing gene (SHOX) are detected in DCO and idiopathic short stature with some rhizomelic body disproportion, whereas mutations in the fibroblast growth factor receptor 3 (FGFR3) gene are found in 40-70% of HCH cases. In this study, we performed mutational analysis of the coding region of the SHOX gene in five DCO and 18 HCH patients, all of whom tested negative for the known HCH-associated FGFR3 mutations. The polymorphic CA-repeat analysis, direct sequencing and Southern blotting were used for detection of deletions and point mutations. The auxological and radiological phenotype of these patients was carefully determined. Three novel mutations in DCO patients were found: (1) a deletion of one base (de1272G) (according to GenBank accession nos. Y11536, Y11535), resulting in a premature stop codon at position 75 of the amino acid sequence; (2) the transversion C485G resulting in the substitution Leu132Val; and (3) the transversion G549T causing an Arg153Leu substitution. These substitutions segregate with the DCO phenotype and affect evolutionarily conserved homeodomain residues, based on a comparison of homeobox containing proteins in 13 species. Moreover, these changes were not found in 80 unrelated, unaffected individuals. This strongly suggests that these mutations are pathogenic. The phenotype of our patients with DCO and HCH varied from mild to severe shortness and body disproportion. These results further support clinical and genetic heterogeneity of dyschondrosteosis and hypochondroplasia.     

Pheromone trap for the eastern tent caterpillar moth

The discovery that the eastern tent caterpillar Malacosoma americanum (F.) causes mare reproductive loss syndrome (MRLS), and thus has the potential to continue to result in major economic losses to the equine industry of Kentucky, has resulted in an intensive effort to identify practical means to monitor and control this defoliator, including these experiments to optimize a sex pheromone trap for this pest. A pheromone-baited delta trap with a large opening, such as InterceptST Delta, was more effective than other tested traps. Orange delta traps caught more moths than other tested colors. ETC males are caught at all tested heights within the tree canopy. For monitoring flights, setting traps at 1.5 m would allow easy counting of moths. A 9:1 blend of (E,Z)-5,7-dodecadienal (ETC-Ald) and (E,Z)-5,7-dodecadienol (ETC-OH) was most effective in capturing males. Increasing loading doses of a 3:1 blend (Ald:OH) resulted in the capture of increasing numbers of moths, but a 9:1 blend was more effective than 3:1 blend even at a nine-fold lower loading rate. Pheromone-impregnated white septa caught more moths than gray septa at the same loading dose. The advantages and limitations of using pheromone traps for monitoring M. americanum are discussed.     

Different defense strategies of Dendrolimus pini, Galleria mellonella, and Calliphora vicina against fungal infection

The resistance of Galleria mellonella, Dendrolimus pini, and Calliphora vicina larvae against infection by the enthomopathogen Conidiobolus coronatus was shown to vary among the studied species. Exposure of both G. mellonella and D. pini larvae to the fungus resulted in rapid insect death, while all the C. vicina larvae remained unharmed. Microscopic studies revealed diverse responses of the three species to the fungal pathogen: (1) the body cavities of D. pini larvae were completely overgrown by fungal hyphae, with no signs of hemocyte response, (2) infected G. mellonella larvae formed melanotic capsules surrounding the fungal pathogen, and (3) the conidia of C. coronatus did not germinate on the cuticle of C. vicina larvae. The in vitro study on the degradation of the insect cuticle by proteases secreted by C. coronatus revealed that the G. mellonella cuticle degraded at the highest rate. The antiproteolytic capacities of insect hemolymph against fungal proteases correlated well with the insects' susceptibility to fungal infection. The antiproteolytic capacities of insect hemolymph against fungal proteases correlated well with the insects' susceptibility to fungal infection. Of all the tested species, only plasmatocytes exhibited phagocytic potential. Exposure to the fungal pathogen resulted in elevated phagocytic activity, found to be the highest in the infected G. mellonella. The incubation of insect hemolymph with fungal conidia and hyphae revealed diverse reactions of hemocytes of the studied insect species. The encapsulation potential of D. pini hemocytes was low. Hemocytes of G. mellonella showed a high ability to attach and encapsulate fungal structures. Incubation of C. vicina hemolymph with C. coronatus did not result in any hemocytic response. Phenoloxidase (PO) activity was found to be highest in D. pini hemolymph, moderate in G. mellonella, and lowest in the hemolymph of C. vicina. Fungal infection resulted in a significant decrease of PO activity in G. mellonela larvae, while that in the larvae of D. pini remained unchanged. PO activity in C. vicina exposed to fungus slightly increased. The lysozyme-like activity increased in the plasma of all three insect species after contact with the fungal pathogen. Anti E. coli activity was detected neither in control nor in infected D. pini larvae. No detectable anti E. coli activity was found in the control larvae of G. mellonella; however, its exposure to C. coronatus resulted in an increase in the activity to detectable level. In the case of C. vicina exposure to the fungus, the anti E. coli activity was significantly higher than in control larvae. The defense mechanisms of D. pini (species of economic importance in Europe) are presented for the first time.     

Interaction between muscle aldolase and muscle fructose 1,6-bisphosphatase results in the substrate channeling

Fructose 1,6-bisphosphatase (FBPase) is known to form a supramolecular complex with alpha-actinin and aldolase on both sides of the Z-line in skeletal muscle cells. It has been proposed that association of aldolase with FBPase not only desensitizes muscle FBPase toward AMP inhibition but it also might enable the channeling of intermediates between the enzymes [Rakus et al. (2003) FEBS Lett. 547, 11-14]. In the present paper, we tested the possibility of fructose 1,6-bisphosphate (F1,6-P(2)) channeling between aldolase and FBPase using the approach in which an inactive form of FBPase competed with active FBPase for binding to aldolase and thus decreased the rate of aldolase-FBPase reaction. The results showed that F1,6-P(2) is transferred directly from aldolase to FBPase without mixing with the bulk phase. Further evidence that F1,6-P(2) is channeled from aldolase to FBPase comes from the experiments investigating the inhibitory effect of a high concentration of magnesium ions on aldolase-FBPase activity. FBPase in a complex with aldolase, contrary to free muscle FBPase, was not inhibited by high Mg(2+) concentrations, which suggests that free F1,6-P(2) was not present in the assay mixture during the reaction. A real-time interaction analysis between aldolase and FBPase revealed a dual role of Mg(2+) in the regulation of the aldolase-FBPase complex stability. A physiological concentration of Mg(2+) increased the affinity of muscle FBPase to muscle aldolase, whereas higher concentrations of the cation decreased the concentration of the complex. We hypothesized that the presence of Mg(2+) stabilizes a positively charged cavity within FBPase and that it might enable an interaction with aldolase. Because magnesium decreased the binding constant (K(a)) between aldolase and FBPase in a manner similar to the decrease of K(a) caused by monovalent cations, it is postulated that electrostatic attraction might be a driving force for the complex formation. It is presumed that the biological relevance of F1,6-P(2) channeling between aldolase and FBPase is protection of this glyconeogenic, as well as glycolytic, intermediate against degradation by cytosolic aldolase, which is one of the most abundant enzyme of glycolysis.     

Morphological parameters of the angiogenic response in pancreatic ductal adenocarcinoma--correlation with histological grading and clinical data.

The aim of this study was to evaluate angiogenesis in tissue of the pancreatic ductal adenocarcinoma and to correlate it with histopathological data such as tumour differentiation, tumour size, lymph node metastasis and patients survival. Tumour samples obtained during surgery from 36 patients were immunostained for the presence of blood vessels with monoclonal antibody against the CD31 molecule. Evaluation of microvasculature was perfomed by counting the microvessel density (MVD) in selected areas under light microscope as well as by computer assisted image analysis (CAIA). In the latter, the following parameters were used for assessment of microvessels: mean number/field, mean area of the vessels, total area/field and total perimeter/field. MVD values obtained under optical microscope and with CAIA were highly correlated. All parameters characterising microvasculature in CAIA also revealed a significant correlation with the histological grading of tumours; generally the less differentiated tumours manifested more extensive vascular network. No significant relationship was found between the tumour size and any of the CAIA parameters. The area of vessels (both total and mean values) revealed a significant, inverse correlation with the incidence of lymph node metastases. The same type of correlation was also found between the mean vessel area and the postoperative survival period. The results show that CAIA of microvessels offers new parameters with some predictive value for the outcome of patients with pancreatic cancer.     

Poly-C specific ribonuclease activity correlates with increased concentrations of IL-6, IL-8 and sTNFR55/sTNFR75 in plasma of patients with acute pancreatitis.

Plasma pancreatic-type Poly-C specific ribonuclease (P-RNase)-enzyme activity increases in patients with acute pancreatitis (AP) who develop pancreatic necrosis and severe disease course. It is considered as a marker of pancreatic tissue destruction. The aim of this study was to estimate interrelations between major inflammatory cytokines such as: interleukin 6 (IL-6), interleukin 8 (IL-8) and tumor necrosis factor soluble receptors: sTNFR55 and sTNFR75 output, and plasma P-RNase activity. The study was carried out in a group of 56 patients with AP, where 20 developed pancreatic necrosis. It was found that serum P-RNase concentration and levels of all studied inflammatory cytokines significantly increase already in the first day from diagnose of the disease (2.5 folds for P-RNase, 20 for IL-8, about 200 for IL-6 and 1.5 for receptors, respectively). In the first day from admission to hospital, P-RNase activity significantly correlated with plasma concentration of studied inflammatory cytokines. The most pronounced correlation was found for P-RNase and IL-6 in days 1-4 from diagnose, manifested by Pearson correlation r coefficients amounting to 0.86, 0.79, 0.60 and 0.57 respectively (p<0.001). Dividing the studied AP patients into two groups, varying in severity of disease a significant differences in P-RNase and IL-6, IL-8 and sTNFR55/sTNFR75 were found. In patients with acute necrotizing pancreatitis P-RNase significantly correlate with levels of major inflammatory cytokines. Carried out studies suggest that activity of P-RNase reflects severity of inflammatory reaction, which is dependent on development of pancreatic injury and tissue necrosis in AP.