KIT and PDGFRalpha mutations in 104 patients with gastrointestinal stromal tumors (GISTs): a population-based study.

BACKGROUND: The prognostic significance of KIT or platelet-derived growth factor receptor alpha (PDGFRalpha) mutations in gastrointestinal stromal tumors (GISTs) is still controversial. PATIENTS AND METHODS: In all, 104 patients were diagnosed with GISTs by KIT immunoreactivity; tumor DNA was sequenced for the presence of mutations in KIT exons 9, 11, 13 and 17 and in PDGFRalpha exons 12 and 18. Disease-free survival (DFS) was analyzed in 85 radically resected patients. RESULTS: KIT mutations occurred in exon 11 (69), in exon 9 (11) and in exon 17 (1). PDGFRalpha mutations were detected in exon 18 (10) and in exon 12 (3). Ten GISTs were wild type. Exon 11 mutations were as follows: deletions in 42 cases and point mutations in 20 cases and insertions and duplications, respectively, in 2 and 5 cases. A better trend in DFS was evident for duplicated and point-mutated exon 11 KIT GISTs. There was a significant association between PDGFRalpha mutations, gastric location and lower mitotic index. Moreover, PDGFRalpha-mutated GISTs seemed to have a better outcome. CONCLUSIONS: Point mutations and duplications in KIT exon 11 are associated with a better clinical trend in DFS. PDGFRalpha-mutated GISTs are preferentially localized in the stomach and seem to have a favorable clinical behavior.

Effectiveness of the CRCAPRO program in identifying patients suspected for HNPCC.

Subjects affected by hereditary non-polyposis colorectal cancer exhibit a high susceptibility to colon and extracolonic tumours, due to MMR gene defects. Revised Bethesda criteria are used to select patients as candidates for genetic tests. Recently, the CRCAPRO model has been developed, based on family history of colorectal and endometrial cancers. Our study aims to evaluate the reliability of CRCAPRO in identifying mutation carriers. We used the CRCAPRO program to evaluate carrier probability risk in 99 patients fulfilling Amsterdam or Bethesda guidelines. MLH1 and MSH2 were studied by direct sequencing in all the 99 patients, and the study of microsatellite instability and of MMR proteins expression was performed. Nine MLH1 and nine MSH2 germline mutations were identified. Five out of the nine patients with MLH1 mutation showed a CRCAPRO risk evaluation of less than 20%. The same happened for four out of nine patients with MSH2 mutation. Of the 17 patients with an estimated risk of more than 80%, only four harboured a mutation, all in the MSH2 gene. The highest risk calculated by the CRCAPRO system in the nine carriers of a MLH1 mutation has been 31.7%. In our experience, the CRCAPRO program sensitivity and specificity appears to be low but needs to be further evaluated in larger samples.

MSH2 splice site mutation and endometrial cancer.

Hereditary nonpolyposis colorectal cancer (HNPCC) is an inherited syndrome of cancer susceptibility caused by germ line mutations of genes participating in mismatch repair (MMR). Carriers of MMR gene mutations have an increased risk of colorectal cancers and cancer of other organs. Tumors of the endometrium represent the most frequent extracolonic malignancies in HNPCC. It has been suggested that women harboring MMR gene mutations have a higher risk of endometrial cancer than of colon cancer. Here, we describe an HNPCC patient with early-onset endometrial cancer and a strong familial history of endometrial tumors who harbored a germ line MSH2 splice site mutation (IVS9_2A>G). This mutation was responsible for abnormal messenger RNA processing, leading to the introduction of a premature stop signal and to the expression of a truncated MSH2 protein. In addition, the same mutation was associated with loss of MSH2 protein expression, high microsatellite instability, and PTEN inactivation. Although a direct relationship between the endometrial cancer susceptibility and the MSH2 mutation we found cannot be established, our observations, consistent with the work of other authors, suggest the involvement of germ line MSH2 abnormalities in endometrial tumor development and support the case for endometrial cancer screening in women from HNPCC families.

A peptide fraction from factor VIII reduces PKC activity in cultured endothelial cells.

A peptide fraction of low molecular weight (Vueffe) prepared from bovine Factor VIII by enzymatic hydrolysis with trypsin, reduces significantly (p<0.05) membrane bound protein kinase C (PKC) activity in cultured bovine pulmonary artery endothelial cells grown with enhanced glucose levels (22.2 mM) or stimulated by phorbol 12-myristate 13-acetate (PMA). The activation of PKC is a common pathway by which mediators increase transendothelial permeability during tissue inflammation and in the development of diabetic vascular complications. Our results suggest that the antihaemorrhagic properties of Vueffe could be related to a decrease in endothelial permeability mediated by PKC.

Hepatotropic conjugate of adenine arabinoside monophosphate with lactosaminated poly-L-lysine. Synthesis of the carrier and pharmacological properties of the conjugate.

BACKGROUND/AIMS: The hepatotropic conjugate of adenine arabinoside monophosphate with lactosaminated poly-L-lysine (L-Poly(Lys)) must have a high solubility in order to be injected in a small volume compatible with the intramuscular route. In this paper the molecular weights of Poly(Lys) which allowed the synthesis of conjugates with the properties of high solubility and limited loss by the kidney were determined and a procedure for obtaining Poly(Lys) preparations with the required range of polymerization has been described. METHODS: Conjugates were prepared using Poly(Lys) of different molecular weights obtained by the procedure described here or purchased from a commercial source. Their solubility and renal loss in mice was determined. RESULTS: Poly(Lys) with molecular weights ranging from 45,000 and 65,000 Da guarantees high solubility and low renal elimination of the conjugates. Conjugate preparations with these properties, intramuscularly administered to woodchuck hepatitis virus-infected woodchucks for 37 days at a daily dose of 5.8 mg/kg exerted a strong antiviral activity. These preparations were devoid of acute toxicity in rat and caused no toxic effects when injected intramuscularly daily for 28 days at a dose ten times higher than that active in woodchucks. CONCLUSIONS: The results support the possibility of a clinical use of L-Poly(Lys) to obtain liver targeting of adenine arabinoside monophosphate for the treatment of chronic hepatitis B virus infection.

New chiral inhibitors of induced platelet aggregation: the enantiomeric specificity of (R)- and (S)-methyl and ethyl esters of 1-(4-[(1-hydroxycarbonyl)-ethoxy]-benzyl)-1H-1,2,3-triazole as a tool for determining their biological target.

The synthesis of title enantiomers was accomplished and their biological behaviour as inhibitors of rabbit platelet aggregation process induced by ADP and arachidonic acid was determined. Structure-activity comparison with that of SM-12502 [(2R,5S)-(+) 3,5-dimethyl-2-(3-pyridyl)-thiazolidin-4-one hydrochloride] and Dazoxiben [4-[2-(1H-imidazol-1-yl)-ethoxy]-benzoic acid] allowed us to formulate the possible capability for the synthesized compounds to interact with the biological targets of the model molecules.

A comparison of the activity of a heparan sulphate of defined molecular weight range (7500-15,000 Da) with heparin and dermatan sulphate.

The fibrinolytic and anticoagulant activities of heparan sulphate (HS) and dermatan sulphate (DS) were compared with those of heparin using in vitro tests. Our results demonstrate that HS has higher profibrinolytic activity than heparin and DS. Although 50 times less potent than heparin in inhibiting factor IIa, HS is three times more active than DS. The action of HS resides in HCH-mediated factor IIa inhibition combined with an ATIII-mediated inhibition. DS has no action on ATIII-mediated inhibition of factor IIa. The comparison of the anticoagulant activities of the three compounds confirmed the very limited anticoagulant effect of both HS and DS in comparison with heparin.

Synthesis, antiinflammatory activity and platelet antiaggregating activity of a new series of beta-aminoxypropionic acids.

A series of beta-aminoxypropionic acids (AOPAs) had previously been designed and synthesised as analogues of antiinflammatory arylacetic acids (ArAAs) in which the Ar portion is substituted by the (methyleneaminoxy) methyl moiety (C = NOCH2, MAOMM). Most of these AOPAs had exhibited a significant antiinflammatory and antiaggregating activity. This paper reports the synthesis of a new series of beta-aminoxypropionic acids (SAOPAs) which include the saturated (methylaminoxy)methyl moiety (CHNH-OCH2, SMAOMM) in the place of the MAOMM present in AOPAs. The antiinflammatory activity of SAOPAs was evaluated by the carrageenan-induced paw edema method and the antiaggregating activity was evaluated by means of tests using arachidonic acid (AA) and adenosine diphosphate (ADP) as the aggregating agents. Two SAOPAs were evaluated for their capacity to inhibit the cyclooxygenase enzyme by measuring the malondialdehyde (MDA) produced by incubation of sodium arachidonate with platelet-rich plasma (PRP). The pharmacological results showed that the saturation of the iminic double bond led to a reduction or even the disappearance of the antiaggreganting activity, whereas it did not induce any evident changes in the antiinflammatory activity. Theoretical studies were carried out in order to compare the conformation and the molecular reactivity of SAOPAs with those of AOPAs.

Disposition of a new heparan sulfate with fibrinolytic activity in the rat.

A fluoresceinated derivative of a new heparan sulfate fraction (HS) was administered by intravenous route to rats. The compound was similar to the unlabelled compound and biologically active. After i.v. injection, pharmacokinetic parameters were analyzed and discussed according to a two-compartment open model. In addition, experiments performed with the unlabelled compound indicate that the HS is absorbed through the intestinal mucosa, reaches the highest plasmatic concentration after 90 min and is partially recovered, unmodified, in urine. Other sets of experiments, in vitro, show that the compound is degraded in the presence of hepatic microsomal preparation while it is not metabolized by plasma, confirming that the liver plays an important role in the metabolism and consequently on the activity of the compound. The overall results are in accordance with the fibrinolytic and antithrombotic activity of HS administered orally to animals and man.

Effects of a new aldose reductase inhibitor on diabetic complications in rats.

The effects of a new aldose reductase inhibitor, 7-fluoro-2-(N-methyl-N-carboxymethyl)sulfamoyl xanthone (BAL-ARI8, CAS 124066-40-6), on the diabetic complications of streptozotocin-induced diabetic rats were studied. The daily administration of BAL-ARI8 throughout the 8-week course of the experiment sharply decreased the sorbitol accumulation in the lens of the diabetic rats. The incidence of cataract formation was also reduced, being detected in only 45% of BAL-ARI8 treated animals, against the 100% of diabetic controls showing cataract after 8 weeks from diabetes onset. On the other hand, the serum glucose levels remained unchanged. In diabetic controls, there was about a 2.5-fold increase of the total protein urinary excretion during the 24 h. Treatment with BAL-ARI8 prevented up to 70% of this increase. Individual protein components were examined by polyacrylamide gel electrophoresis and quantitated by laser densitometric analysis. Diabetic-induced proteinuria primarily resulted from excretion of newly detected proteins with molecular weight in the range 30,000-60,000 D, together with an increase of albumin (25% of the total excretion) and the presence of new higher molecular weight proteins (greater than 66,000 D). BAL-ARI8 administration resulted in a shift of the protein profile back toward normality i.e. 73% of proteins with molecular weight below 30,000 D, 7.5% albumin and no proteins above 66,000 D. These results suggest that BAL-ARI8 may represent a therapeutic approach for the management of diabetic complications.

Some chemical and biological properties of the low molecular weight heparan sulfate alpha-idosane.

A low molecular weight heparan sulfate derivative, alpha-idosane was separated from a mixture of glycosaminoglycans extracted from porcine mucosa. Its molecular weight, sulfur, uronic acid and hexosamine contents, C-NMR spectrum and electrophoretic properties are reported in this paper. The pharmacological effects of a-idosane were investigated "ex vivo" in dogs and rats. At doses of 10-50 mg/kg p.o., a-idosane shows fibrinolytic activity but it is devoid of anticoagulant action. At the dose of 100 mg/kg p.o. a-idosane exertes a significant anti-inflammatory effect but is unable to protect the rats against arachidonate-induced sudden death.

"In vitro" fibrinolytic activity of a new low molecular weight heparan sulfate.

The fibrinolytic activity, effect on the fibrinolytic activity of plasmin, anticoagulant activity and anti platelet aggregation activity of a low molecular weight heparan sulfate (LMW HS Bal) were investigated "in vitro" on blood plasma obtained from rats and rabbits. LMW HS Bal at concentrations as low as 0.25-2 micrograms/ml prevented thrombin-induced platelet aggregation. At concentrations 25 to 50 times larger it showed no significant anticoagulant activity but a marked fibrinolytic effect. At still larger concentrations LMW HS Bal also potentiated the fibrinolytic activity of plasmin. Conversely, unfractionated heparan sulfate (UHS) at concentrations of 25 to 50 micrograms/ml, only showed anticoagulant activity with no fibrinolytic activity.

On the effects of tranexamic acid and its isobenzedrine ester on plasminogen activation and streptokinase induced fibrinolysis.

A comparison between the inhibitory capability of Tranexamic acid (AMCA) and its isobenzedrine ester (IB-AMCA) on the streptokinase and urokinase induced plasminogen activation, indicated in vitro a higher potency of the ester derivative. A peculiar activatory rather than inhibitory effect on the plasminogen activation was exerted by AMCA and aminocaproic acid at relatively low concentrations. Attempts to show in vivo the in vitro observed differences between AMCA and IB-AMCA action are reported.

Polypeptide fraction from bovine factor VIII does not influence human platelet aggregation and blood coagulation.

It is well known that high molecular weight bovine factor VIII is able to aggregate human platelets and possesses procoagulant activities. There is also growing body of evidence that the hydrolysis of bovine factor VIII abolishes its aggregating and coagulative properties. We have shown in this paper that a polypeptide fraction (molecular weight 1000-25000 daltons) from bovine factor VIII does not aggregate platelets nor affect blood coagulation. In this study we investigate the action of the polypeptide fraction derived from bovine factor VIII and suggest that its effect may occur only at endothelium level without an involvement of platelets as well as blood coagulation.

Induction of phosphoribomutase in Bacillus cereus growing on nucleosides.

In this paper we show that phosphoribomutase is induced in Bacillus cereus by the same metabolizable purine and pyrimidine ribonucleosides previously shown to induce the purine nucleoside phosphorylase (Tozzi, M.G., Sgarrella, F. and Ipata, P.L. (1981) Biochim. Biophys. Acta 678, 460-466). The mutase allows ribose 1-phosphate formed from nucleosides to be utilized by the cell through the pentose cycle, upon transformation to ribose 5-phosphate. The equilibrium constant of the mutase reaction is towards ribose-5-phosphate formation. The coordinate induction of the two enzymes completes the picture of the molecular events leading to the utilization of the sugar moiety of purine nucleotides and nucleosides as an energy source (Mura. U., Sgarrella, F. and Ipata, P.L. (1978) J. Biol. Chem. 253, 7905-7909).

Preliminary characterization of adenosine deaminase from Bacillus cereus.

Adenosine deaminase from Bacillus cereus is quite unstable, similarly to other bacterial deaminases, but it shows a peculiar stabilizing effect by some monovalent cations. These include K+, Li+, NH4+ and to a lesser extent Cs+. Maximal stabilization of the deaminase is exerted by K+ at concentrations higher than 20 mM. The enzyme can be rapidly inactivated by sulphydryl reagents such as p-hydroxymercuribenzoate. Since adenosine deaminase from B. cereus, in addition to monovalent cations, is stabilized also by dithiothreitol, a possible influence of monovalent cations on the reactivity of some sulphydryl groups on the enzyme has been suggested.