Novel quinazoline-quinoline alkaloids with cytotoxic and DNA topoisomerase II inhibitory activities

Two new synthetic analogues of luotonins A and F, 7-acetylaminoluotonin A (6) and 3-[3H(quinazolino-4-one)]quinoline (7) were synthesized. The new analogues, along with four natural quinazoline-quinoline alkaloids, luotonins A (1), B (2), E (3), F (4) and a synthetic deoxoluotonin F (5), showed cytotoxic activity (IC(50) 1.8-40.0 microg/mL) and DNA topoisomerase II inhibition at a concentration of 25 microM.     

Tetrahydroisoquinoline based sulfonamide hydroxamates as potent matrix metalloproteinase inhibitors

The synthesis and MMP inhibitory activity of a series of tetrahydroisoquinoline based sulfonamide hydroxamates are described. In nine MMPs tested, most of the compounds display potent inhibition activity except for MMP-7. Some subtle isozyme selectivity is observed by varying the substituents at the 6- and 7-positions and aromatic ring of arylsulfonyl groups.     

Cold-shock induced high-yield protein production in Escherichia coli

Overexpression of proteins in Escherichia coli at low temperature improves their solubility and stability. Here, we apply the unique features of the cspA gene to develop a series of expression vectors, termed pCold vectors, that drive the high expression of cloned genes upon induction by cold-shock. Several proteins were produced with very high yields, including E. coli EnvZ ATP-binding domain (EnvZ-B) and Xenopus laevis calmodulin (CaM). The pCold vector system can also be used to selectively enrich target proteins with isotopes to study their properties in cell lysates using NMR spectroscopy. We have cloned 38 genes from a range of prokaryotic and eukaryotic organisms into both pCold and pET14 (ref. 3) systems, and found that pCold vectors are highly complementary to the widely used pET vectors.     

Prevalence of the C282Y, H63D, and S65C mutations of the HFE gene in 1,146 newborns from a region of Northern Spain

In Spain, 85% of patients with genetic hemochromatosis (GH) are homozygous for the C282Y mutation of the HFE gene. H63D and S65C mutations of HFE may also play some role in the disease. The aim of this study was to establish the prevalence of C282Y, H63D, and S65C mutations of the HFE gene in newborns in Catalonia, Spain. One thousand one hundred forty-six newborn screening cards were selected randomly. DNA from these cards was extracted and HFE mutations were analyzed with the LightCycler equipment (Roche Diagnostics Gmbh, Mannheim, Germany). Sufficient DNA sample was obtained to screen for the three mutations in 1,043 cases (91%). The allelic frequencies of C282Y, H63D, and S65C mutations were 0.03 (IC 95% 0.022-0.037), 0.2 (IC 95% 0.19-0.22), and 0.01 (95% confidence interval [CI] 0.006-0.015), respectively. The frequency of C282Y homozygous newborns was 0.001 (95% CI 0.0005-0.0014). The frequencies of newborns doubly heterozygous for C282Y/H63D and C282Y/S65C were 0.01 (95% CI 0.005-0.02) and 0.002 (95% CI 0.0002-0.01), respectively. The allelic frequency of C282Y mutation is similar to that observed in Southern France, in the Czech Republic and in some areas of Italy. The allelic frequency of H63D mutation in Catalonia is the highest reported to date. Nevertheless, S65C is infrequent. These data should be kept in mind when designing hemochromatosis genotypic screening programs in Catalonia.     

An accumulation of insulin-like growth factor I (IGF-I) in human myometrium and uterine leiomyomas in various stages of tumour growth

It is commonly thought that uterine leiomyomas result from hyperstimulation of myometrium by ovarian hormones. Some observations suggest that cytokines and growth factors are intermediate elements through which the ovarian hormones may exert their growth-stimulatory effects on leiomyomas. Human myometrium and uterine leiomyomas of various weights were homogenised and extracted with 1 M acetic acid or with 0.05 M Tris/HCl, pH 7.6. The extracts were assayed for IGF-I using the ELISA technique. It was found that 0.05 M Tris/HCl extracts contained several times more IGF-I than the 1 M acetic acid extracts. Nanogram amounts of IGF-I were found in both control myometrium and in leiomyomas. It was found that the amounts of IGF-I extracted from leiomyomas were distinctly higher in comparison to control myometrium and they increased as a function of tumour growth. Polyacrylamide gel electrophoresis, followed by Western immunoblotting, demonstrated that IGF-I in acidic and alkaline extracts exists as stable complexes, probably with extracellular matrix components. No free IGF-I was detected. Furthermore, it was found that some components of both the acidic and alkaline extracts were able to bind exogenous (125)I-labeled IGF-I. It is suggested that IGF-I plays an important role both in myometrium biology and in the growth of uterine leiomyomas.     

Genotoxicity of inhalation anaesthetics: DNA lesions generated by sevoflurane in vitro and in vivo

A moderate genotoxic activity of halothane and isoflurane applied as volatile anaesthetics has already been shown. The aim of this work was to estimate a potential genotoxicity of sevoflurane, introduced to clinical practice later than halothane and isoflurane. A genotoxic activity of all three compounds was estimated by using the comet assay in human peripheral blood lymphocytes (PBL) proliferating in vitro. We demonstrated that in contrast to the previously studied anaesthetics, sevoflurane did not induce any increase in DNA migration in the studied conditions. To estimate a genotoxic effect of a prolonged exposure to halogenated anaesthetics in vivo, PBL taken from operating room personnel (n = 29) were tested for DNA degradation and compared with those from a control non-exposed group (n = 20). No significant differences were detected between the groups. We conclude that sevoflurane does not have genotoxic properties, both in vitro and in vivo.     

Cloning a DNA marker associated to wheat scab resistance

Wheat head blight caused mainly by Fusarium graminearum, is an important wheat disease, causing yield and quality losses. The breeding of resistant varieties is the key measure to control this disease, but the conventional breeding method is of low efficiency. The marker-assisted selection (MAS) can significantly improve the breeding efficiency. In this study, four RAPD (randomly amplified polymorphic DNA) markers linked to FHB resistance were obtained and one was cloned and sequenced. F7 recombinant inbred lines (RILs) were derived from the F1 of the cross Ning894037 (resistant)/Alondra (susceptible) by the single-seed descent method. Scab resistance of F7 RILs was evaluated in the greenhouse by injecting conidiospores into a central floret. Scab symptoms were evaluated on the 21st day after inoculation. Disease severity was assessed as the percentage of infected spikelets/spike. The F7 RIL population displayed a normal distribution, transgressive segregation and significant variation for FHB severity. DNA from resistant and susceptible parents was analyzed with 520 RAPD primers. Four markers (S1384-640,S1360-600, S1319-350,S1319-820) linked to FHB resistance were obtained. DNA of S1384-640 was recovered, subjected to re-amplification by using S1384 primer and the same protocol as for RAPD analysis and identified the rightness. The PCR product of S1384-640 was ligated into the pUCm-T vector, and cloned into fresh competent cells of Escherichia coli strain DH5alpha RAPD analysis showed that the inserts of the recombinant plasmids were DNA of S1384-640. The sequencing result showed that the cloned fragment was 648 bp.     

The role of ephrins and Eph receptors in cancer

Eph receptors are the largest receptor tyrosine kinase family of transmembrane proteins with an extracellular domain capable of recognizing signals from the cells’ environment and influencing cell–cell interaction and cell migration. Ephrins are the ligands to Eph receptors and stimulate bi-directional signaling of the Eph/ephrin axis. Eph receptor and ephrin overexpression can result in tumorigenesis as related to tumor growth and survival and is associated with angiogenesis and metastasis in many types of human cancer. Recent data suggest that Eph/ephrin signaling could play an important role in the development of novel inhibition strategies and cancer treatments to potentially target this receptor tyrosine kinase and/or its ligand. A deeper understanding of the molecular basis for normal versus defective cell–cell interaction through the Eph/ephrin axis will enable the potential development of novel cancer treatments. This review emphasizes the biology of Eph/ephrin as well as the potential for novel targeted therapy through this pathway.     

Early anti-apoptosis treatment reduces myocardial infarct size after a prolonged reperfusion

OBJECTIVE : Significant myocardial apoptosis occurs in ischemia/reperfused hearts. However, the contribution of apoptosis to the development of myocardial injury remains controversial. The present study attempted to obtain evidence that inhibition of apoptosis at early reperfusion can reduce myocardial infarction after prolonged reperfusion. METHODS : Adult male rats were subjected to 30 min ischemia and 4 (apoptosis assay) or 24 h (myocardial infarction determination) of reperfusion and treated with vehicle, SB 239063, insulin or insulin plus wortmannin. RESULTS : Treatment with SB 239063 or insulin markedly decreased myocardial apoptosis (10.6 +/- 1.5% and 7.9 +/- 0.9% respectively, P < 0.01 vs. vehicle) and significantly reduced infarct size (43 +/- 3.6% and 35 +/- 2.9%, respectively, P < 0.01 vs. vehicle). Most interestingly, inhibition of insulin signaling with wortmannin to block insulin signaling not only blocked insulin's anti-apoptotic effect, but also abolished its infarct reduction property. CONCLUSION : These data indicate that apoptosis contributes to the development of myocardial infarction, and inhibition of apoptosis at early reperfusion reduces the myocardial infarction.     

Functional assessment of cryopreserved pig aortas for pharmaceutical research

Several in vitro studies have demonstrated diminished post-thaw functional activity. Therefore, the aim of this study was to investigate the consequences of thawing and storage method used on the post-thaw functional activity of cryopreserved pig aortas with the aim of adjusting the freezing and thawing protocol so that the vascular segments are preserved in the best possible state, maintaining structure and functionality so that they can later be transplanted with success. In vitro responses of frozen, thawed pig aortas were used to investigate the functional activity after thawing at 15 degrees C and 100 degrees C/min and after storage in gas or liquid phase of liquid nitrogen. Cryopreservation was performed in RPMI 1640 medium + 10% dimethylsulfoxide and the rate of cooling was -1 degrees C/min, until -150 degrees C was reached.After thawing the maximal contractile responses to all the contracting agonists tested (KCl, noradrenaline) were in the ranges of 13-27% compared with the responses in unfrozen pig aortas. Contractile responses were slightly better when thawing was performed at 15 degrees C/min compared with 100 degrees C/min. The endothelium independent relaxant responses to sodium nitroprusside were reduced ( P < 0.05). Cryostorage of pig arteries also resulted in a loss of the endothelium-dependent relaxant response to acetylcholine. The cryopreservation method used provided a limited preservation of pig aorta contractibility, a reduction of the endothelium independent relaxant responses, and no apparent preservation of the endothelium-dependent relaxation. It is possible that further refinements of the cryopreservation protocol might allow better post-thaw functional recovery of pig aortas.