Swarming and mating activity of Anopheles gambiae mosquitoes in semi-field enclosures.

Anopheles gambiae Giles sensu stricto (Diptera: Culicidae) is the major Afro-tropical vector of malaria. Novel strategies proposed for the elimination and eradication of this mosquito vector are based on the use of genetic approaches, such as the sterile insect technique (SIT). These approaches rely on the ability of released males to mate with wild females, and depend on the application of effective protocols to assess the swarming and mating behaviours of laboratory-reared insects prior to their release. The present study evaluated whether large semi-field enclosures can be utilized to study the ability of males from a laboratory colony to respond to natural environmental stimuli and initiate normal mating behaviour. Laboratory-reared males exhibited spatiotemporally consistent swarming behaviour within the study enclosures. Swarm initiation, peak and termination time closely tracked sunset. Comparable insemination rates were observed in females captured in copula in the semi-field cages relative to females in small laboratory cages. Oviposition rates after blood feeding were also similar to those observed in laboratory settings. The data suggest that outdoor enclosures are suitable for studying swarming and mating in laboratory-bred males in field-like settings, providing an important reference for future studies aimed at assessing the comparative mating ability of strains for SIT and other vector control strategies.

Glyoxalase I inhibition induces apoptosis in irradiated MCF-7 cells via a novel mechanism involving Hsp27, p53 and NF-κB.

BACKGROUND: Glyoxalase I (GI) is a cellular defence enzyme involved in the detoxification of methylglyoxal (MG), a cytotoxic byproduct of glycolysis, and MG-derived advanced glycation end products (AGEs). Argpyrimidine (AP), one of the major AGEs coming from MG modifications of proteins arginines, is a pro-apoptotic agent. Radiotherapy is an important modality widely used in cancer treatment. Exposure of cells to ionising radiation (IR) results in a number of complex biological responses, including apoptosis. The present study was aimed at investigating whether, and through which mechanism, GI was involved in IR-induced apoptosis. METHODS: Apoptosis, by TUNEL assay, transcript and protein levels or enzymatic activity, by RT-PCR, western blot and spectrophotometric methods, respectively, were evaluated in irradiated MCF-7 breast cancer cells, also in experiments with appropriate inhibitors or using small interfering RNA. RESULTS: Ionising radiation induced a dramatic reactive oxygen species (ROS)-mediated inhibition of GI, leading to AP-modified Hsp27 protein accumulation that, in a mechanism involving p53 and NF-κB, triggered an apoptotic mitochondrial pathway. Inhibition of GI occurred at both functional and transcriptional levels, the latter occurring via ERK1/2 MAPK and ERα modulation. CONCLUSIONS: Glyoxalase I is involved in the IR-induced MCF-7 cell mitochondrial apoptotic pathway via a novel mechanism involving Hsp27, p53 and NF-κB.

Changes in CSF acetyl- and butyrylcholinesterase activity after long-term treatment with AChE inhibitors in Alzheimer's disease.

OBJECTIVES: To measure cerebrospinal fluid (CSF) activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in patients with Alzheimer's disease (AD) participating in randomized clinical trials from three European centers, before and after long-term treatment with different AChE inhibitors (AChEIs). MATERIALS AND METHODS: Of the 144 patients included in the study, 104 were treated with donepezil, 15 with galantamine, 16 with rivastigmine, and nine with placebo. CSF AChE and BChE activities were measured at baseline and after 1- year treatment. RESULTS: Donepezil and galantamine groups showed a significant increase in CSF AChE activity at follow-up, while no changes for BChE activity were observed; in donepezil group, a positive correlation between plasma concentration and AChE activity was documented. Conversely, in rivastigmine group, a decrease in CSF activity of both enzymes was observed. CSF AChE and BChE activities were not correlated with the clinical outcome in any group considered. CSF biomarkers did not show any change after treatment. CONCLUSIONS: AChEIs differently influence the activity of target enzymes in CSF independent of their pharmacodynamic effects.

[Prolonged exposure to crystalline silica Min-U-Sil-5 influences apoptosis or extracellular matrix genes expression in human bronchial epithelial cells]. / L'esposizione prolungata alla silice cristallina Min-U-Sil-5 influenza l'espressione di geni della matrice extracellulare o coinvolti nell'apoptosi in cellule epiteliali bronchiali umane BEAS-2B.

Crystalline silica (Min-U-Sil-5) induces oxidative stress in human bronchial epithelial cells (BEAS-2B), through the intracellular accumulation of ROS that cause oxidative damage leading to the degradation of extracellular matrix (ECM) proteins and to the loss of cell adhesion molecules inducing apoptosis and genotoxic damage. This paper briefly summarizes some of the recent findings from our laboratories with emphasis on the molecular events by which the cronic and cumulative exposure to crystalline silica can induce cellular damage that promotes changes in extracellular matrix and in apoptosis gene expression.

Female reproductive dysfunction during ageing: role of methylglyoxal in the formation of advanced glycation endproducts in ovaries of reproductively-aged mice.

Reproductive dysfunction with ageing has been so far extensively characterized in terms of depletion of ovarian follicles and reduced ability to produce gametes competent for fertilization. Nevertheless, molecular mechanisms underlying this process are still poorly understood. In the present study we addressed the hypothesis that methylglyoxal (MG), a major precursor of Advanced Glycation Endproducts (AGE), may contribute to molecular damage occurring during ovarian ageing. Our results showed that the biochemical activity of glyoxalase 1, the main component of the MG scavenging system, is significantly decreased in ovaries from reproductively-aged mice in comparison with the young group. This effect was associated with decreased expression at protein and RNA level of this enzyme and increased intraovarian level of MG. MG-arginine adducts argpyrimidine as detected with a specific antibody was found to accumulate with ageing in specific ovarian compartments. Separation of ovarian proteins by 2D gels and Western blotting revealed an approximate 30-fold increase in the extent of protein glycation in aged ovaries along with the appearance of eight argpyrimidine modified proteins exclusive for the aged group. In conclusion, the present results show that impaired MG detoxification causing relevant damage to the ovarian proteome might be one of the mechanisms underlying reproductive ageing and/or ageing-like ovarian diseases.

[Biomarkers of prostate cancer. A review]. / Biomarker della neoplasia prostatica. Una review.

Prostate cancer has the highest tumour incidence in the male population and represents 9.2% of cancer-related deaths. The most commonly used screening technique up to the present has been serum measurement of PSA which has led to a marked increase in the number of prostate cancer cases diagnosed every year. Nevertheless PSA in the early diagnosis of prostate cancer has many limitations. It can lead to a very high number of unnecessary biopsies in patients with benign prostate hyperplasia and, in addition, may also lead to an overdiagnosis and overtreatment of clinically insignificant neoplasias. Moreover many neoplasias are already present with PSA within normal limits. It is clear, therefore, that new biomarkers for the diagnosis and follow-up of prostate cancer have to be developed. We present a review of the literature in which we have analysed the most promising biomarkers in terms of sensitivity and diagnostic specificity for prostate cancer and which are currently under study, analysing recent developments and future prospects.

[Crystalline silica can induce oxidative stress by inhibiting glyoxalase system in bronchial epithelial cells]. / La silice cristallina (Min-U-Sil 5) induce stress ossidativo inibendo l'espressione del sistema delle gliossalasi in cellule bronchiali umane (BEAS-2B).

UNLABELLED: Chronic inflammation and reactive oxygen species (ROS) production induced by crystalline silica are involved in the development of silicosis and lung cancer pathogenesis. ROS can generate lipid peroxydation of cell membranes that can produce methylglyoxal (MG), a strong cell proliferation inhibitor and apoptosis inducer. MG is naturally removed by glyoxalase I (GI) and glyoxalase II (GII) through a glutathione (GSH) dependent mechanism. Therefore mRNA expression of glyoxalases is correlated to MG concentration and oxidative stress. OBJECTIVES: evaluate oxidative stress induced by crystalline silica by glyoxalases mRNA expression and methylglyoxal concentration MATERIAL AND METHODS: In bronchial epithelial cell culture (BEAS-2B), exposed to 50 microg/cm2 crystalline silica (Min-U-Sil 5), for 2, 6, 12, and 24 hours, GI and GII mRNA levels and MG intracellular concentration were measured respectively by Real-Time PCR and HPLC. RESULTS: Crystalline silica exposure induced a significant reduction in mRNA expression of glyoxalases and an increase of MG intracellular concentration. CONCLUSIONS: The results suggest a possible use of MG and mRNA expression of GI and GII as crystalline silica induced oxidative stress indicators.

Demonstration of glyoxalase II in rat liver mitochondria. Partial purification and occurrence in multiple forms.

Glyoxalase II (S-(2-hydroxyacyl)glutathione hydrolase, EC 3.1.2.6), which has been regarded as a cytosolic enzyme, was also found in rat liver mitochondria. The mitochondrial fraction contained about 10-15% of the total glyoxalase II activity in liver. The actual existence of the specific mitochondrial glyoxalase II was verified by showing that all of the activity of the crude mitochondrial pellet was still present in purified mitochondria prepared in a Ficoll gradient. Subfractionation of the mitochondria by digitonin treatment showed that 56% of the activity resided in the mitochondrial matrix and 19% in the intermembrane space. Partial purification of the enzyme (420-fold) was also achieved. Statistically significant differences were found in the substrate specificities of the mitochondrial and the cytosolic glyoxalase II. Electrophoresis and isoelectric focusing of either the crude mitochondrial extract or of the purified mitochondrial glyoxalase II resolved the enzyme activity into five forms with the respective pI values of 8.1, 7.5, 7.0, 6.85 and 6.6. Three of these forms (pI values 7.0-6.6) were exclusively mitochondrial, with no counterpart in the cytosol. The relative molecular mass of the partially purified enzyme, as estimated by Superose 12 gel chromatography, was 21,000. These results give evidence for the presence of mitochondrial glyoxalase II which is different from the cytosolic enzymes in several characteristics.

Purification and characterization of two forms of glyoxalase II from the liver and brain of Wistar rats.

Glyoxalase II (S-(2-hydroxyacyl)glutathione hydrolase, EC 3.1.2.6) was purified to homogeneity and separated into two forms (alpha, pI = 8.0; beta, pI = 7.4) from both liver and brain of wistar rats by column isoelectric focusing. These forms were also found to have different electrophoretic mobilities. No significant differences were found between the alpha and beta forms from either source in the relative molecular mass (about 24,000) or in Km values using three substrates. The temperature-inactivation profiles were also similar, the two forms being stable up to 50 degrees C. Chemical modification studies with phenylglyoxal suggest that these enzyme forms probably contain arginine residues near the active site. Inactivation of alpha and beta forms by diethylpyrocarbonate and by photooxidation with methylene blue, and protection by S-D-mandeloylglutathione, a slowly reacting substrate, suggest the presence of histidine at the active site. The alpha and beta forms show different half-life values in inactivation by histidine reagents, which may be due to a difference in the active-site structures of these enzymes. The results probably indicate distinct structures (sequences) for alpha and beta forms.

Isolation of glyoxalase II from two different compartments of rat liver mitochondria. Kinetic and immunochemical characterization of the enzymes.

Two separate pools of glyoxalase II were demonstrated in rat liver mitochondria, one in the intermembrane space and the other in the matrix. The enzyme was purified from both sources by affinity chromatography on S-(carbobenzoxy)glutathione-Affi-Gel 40. From both crude and purified preparations polyacrylamide gel-electrophoresis resolved multiple forms of glyoxalase II, two from the intermembrane space and five from the matrix. Among the thioesters of glutathione tested as substrates, S-D-lactoylglutathione was hydrolyzed most efficiently by the enzymes from both sources. Significant differences were observed in the specificities between the intermembrane space and matrix enzymes with S-acetoacetylglutathione, S-acetylglutathione, S-propionylglutathione and S-succinylglutathione as substrates. Pure glyoxalase II from rat liver cytosol was chemically polymerized and used as antigen. Antibodies were raised in rabbits and the antiserum was used for comparison of the two purified mitochondrial enzymes with cytosolic glyoxalase II by immunoblotting. The enzyme purified from the intermembrane space cross-reacted with the antiserum, but the matrix glyoxalase II did not. The results give evidence for the presence in rat liver mitochondria of two species of glyoxalase II with differing characteristics. Only the enzyme from the intermembrane space appears to resemble the cytosolic glyoxalase II forms.

Characterization of a null mutation in ace-1, the gene encoding class A acetylcholinesterase in the nematode Caenorhabditis elegans.

Two genes (ace-1 and ace-2) encode two major classes (A and B) of acetylcholinesterase (AChE) in the nematode Caenorhabditis elegans. A null mutation in ace-1 (allele p1000) suppresses all acetylcholinesterase activity of class A. We have identified an opal mutation TGG (W99)-->TGA (Stop) as the only alteration in the mutated gene. This leads to a truncated protein (98 instead of 620 amino acids) with no enzymatic activity. The mutation also reduces the level of ace-1 transcripts to only 10% of that in wild-type animals. This most likely results from a destabilization of mRNA containing the nonsense message. In contrast, compensation of class B by class A AChE in the null mutant strain ace-2 takes place with unchanged ace-1 mRNA level and enzymatic activity similar to class A AChE.

Acetylcholinesterase in Alzheimer's disease.

Since the discovery of the cholinergic deficit in Alzheimer disease (AD), acetylcholinesterase (AChE) has been widely investigated in tissues involved in the disease. These studies showed modifications in AChE activity and changes in its polymorphism in brain as well as in cerebro-spinal fluid (CSF) and blood. The co-localization of the enzyme in the senile plaque provided evidence of its anomalous features. It has been also shown that AChE forms a stable complex with senile plaque components through its peripheral anionic site. Moreover, the neurotoxicity of amyloid components is increased by the presence of AChE. The occurrence of an altered glycosylation of some AChE forms in AD is closely related to the presence of amyloid formations. Literature on expression, relationships and modifications in the molecular polymorphism of AChE, in brain, CSF and blood in AD is reviewed.

Differing expression of enzymes of the glyoxalase system in superficial and invasive bladder carcinomas.

This work aimed to study the activities of the glyoxalase system enzymes (glyoxalase I (GI) and glyoxalase II (GII) and their gene expression in human bladder carcinomas compared with the corresponding normal mucosa. Samples of these tissues were collected from 26 patients with superficial (SBC) or invasive bladder cancer (IBC) and used to evaluate enzyme activity and gene expression by northern blot analysis. In keeping with the electrophoretic pattern and the expression level of the respective genes, GI activity significantly increased in SBC samples, while it remained unchanged in IBC samples compared with the normal mucosa. In contrast, GII showed a higher activity in the tumour (either SBC or IBC samples) versus normal tissues. These results confirm the role of the glyoxalases in detoxifying cytotoxic methylglyoxal (MG) in bladder cancer. The differing levels of GI activity level and gene expression of GI between the SBC and IBC samples could help in their differential diagnosis.

Acetylcholinesterase in Spirographis spallanzanii (Polychaeta: Sedentaria): presence of two dimeric membrane-bound forms.

In the annelid polychaete Spirographis spallanzanii two acetylcholinesterases, named DS and HSDS, were detected. They differ in relative amount, membrane anchoring and pharmacological properties. Studies with inhibitors evidenced complete inhibition of both acetylcholinesterases by 10(-3) M eserine and different sensitivities for edrophonium or procainamide. Both enzymes, sensitive to BW284c51, were unaffected by iso-OMPA; at variance, only the HSDS form underwent excess-substrate inhibition. DS and HSDS enzymes were solubilized by homogenization in a low-salt or high-salt-Triton X-100 buffer and then purified by affinity chromatography on edrophonium- or procainamide-Sepharose column respectively. According to gel-filtration chromatography, sedimentation analysis and SDS-PAGE, the least represented (30%) DS form is a G2 amphiphilic globular dimer (124-130 kDa, 6.0-7.0S) with S-S linked monomers (66 kDa). Phosphatidylinositol anchors give cell membrane insertion, self-aggregation and detergent (Triton X-100, Brij 97) interaction. The prevailing (70%) HSDS acetylcholinesterase is once again a G2 form similar to DS enzyme in its molecular size (117-125 kDa), sedimentation coefficient (6.0S) of the native form and presence of S-S linked subunits (66 kDa). However, it is likely attached to the cell membrane by involvement of strong electrostatic interactions. DS acetylcholinesterase displays moderate active site specificity with differently sized substrates. The HSDS form is inactive on butyrylthiocholine. DS and HSDS forms show a comparable catalytic efficiency (kcat/K(m)) approaching that of other invertebrate enzymes. The results suggest that DS and HSDS enzymes, likely encoded by distinct genes, are both functional in cholinergic synapses.

Synthesis and study of thiocarbonate derivatives of choline as potential inhibitors of acetylcholinesterase.

Fourteen alkyl and aryl thiocarbonate derivatives of choline were synthesized and studied as potential inhibitors of acetylcholinesterase (AChE). Twelve of the compounds inhibited AChEs derived from calf forebrain, human red blood cells, and octopus brain ranging from low to moderately high inhibition potency. The concentration of each inhibitory compound giving 50% inhibition of enzyme activity (IC50 values, which ranged from 1 x 10(-2) to 8 x 10(-7) M) was determined and is reported; inhibitor constants (Ki values) for the most inhibitory compounds, (1-pentylthiocarbonyl)choline chloride and (1-heptylthiocarbonyl)choline chloride, were calculated from kinetic data and are also reported. The inhibitors are competitive with substrate, and they are not hydrolyzed by the AChE activities. Certain of these new compounds may provide direction for the development of new drugs that have anticholinesterase activity and may be used for the treatment of Alzheimer's disease.

Stimulating effect of ascorbic acid on ascites tumor cell multiplication in vitro.

In a preceding report evidence has been presented that ascorbic acid (AA) accelerated solid tumor growth obtained by inoculating tumor ascites cells into the subcutaneous dorsal region of inbred Balb/cf/Had/Se substrain mice. The present work investigates whether this effect is the result of a direct action of AA on neoplastic cell multiplication or of an indirect action on the tumor cells mediated by changes induced by AA in the organism bearing the tumor. The results obtained demonstrate that AA at low doses (microgram 10-30/ml) stimulates ascites tumor cell multiplication in vitro, while at high doses (microgram 60-120/ml) the opposite occurs. These results suggest that the major growth of solid tumors observed in mice treated with AA may be attribute to the capacity of this substance to favor the multiplication of ATP cells which were used to induce the tumors.

Effects of ascorbic and dehydroascorbic acid on the multiplication of tumor ascites cells in vitro.

The effects of AA and DHA on ATP C+ cell multiplication in vitro were studied by measuring incorporation of 3H thymidine into DNA. The results obtained demonstrate that both AA and DHA have the same effects: they favor cell multiplication at low doses and inhibit it at high doses. Experiments carried out with serial doses of both these substances revealed that AA is more efficient in determining both stimulating and inhibiting effects. The lesser efficiency of DHA may be attributed to its limited stability in culture medium. Studies on the effect of high doses of AA and DHA added to the culture medium in single or fractionated doses revealed that fractionated administration is more efficient in inhibiting cell multiplication than single administration.

Acetylcholinesterase in tentacles of Octopus vulgaris (Cephalopoda). Histochemical localization and characterization of a specific high salt-soluble and heparin-soluble fraction of globular forms.

Transverse sections of Octopus tentacles were stained for acetylcholinesterase (AChE) activity. An intense staining, that was suppressed by preincubation in 10(-5) M eserine, was detected in a number of neuronal cells, nerve fibres and neuromuscular junctions of intrinsic muscles of the arm. Octopus acetylcholinesterase was found as two molecular forms: an amphiphilic dimeric form (G2) sensitive to phosphatidylinositol phospholipase C and a hydrophilic tetrameric (G4) form. Sequential solubilization revealed that a significant portion of both G2 and G4 forms was recovered only in a high salt-soluble fraction (1 M NaCl, no detergent), Heparin (2 mg/ml) was able to solubilize G2 and G4 forms with the same efficiency than 1 M NaCl. The solubilizing effect of heparin was concentration-dependent and was reduced by protamine (2 mg/ml). This suggests that heparin operates through the dissociation of ionic interactions existing in situ between globular forms of AChE and cellular or extracellular polyanionic components. Interaction of AChE molecular forms with heparin has been reported so far in only a few instances and its physiological meaning is uncertain. G2 and G4 forms, interacting or not with heparin, all belong to a single pharmacological class of AChE. This suggests the existence of a single AChE gene. Amphiphilic and hydrophilic subunits thus likely result either from the processing of a single AChE transcript by alternative splicing (as in vertebrate AChE) or from a post-translation modification of a single catalytic peptide.

Acetylcholinesterase at high catalytic efficiency and substrate specificity in the optic lobe of Eledone moschata (Cephalopoda: Octopoda): biochemical characterization and histochemical localization.

In the optic lobe of the cephalopod mollusc Eledone moschata, two acetylcholinesterase forms I and II were detected, both showing a marked active site specificity with differently sized substrates. Catalytic efficiency (kcat/Km) of the prevailing form II is similar to that of acetylcholinesterases from vertebrate nervous system. Enzyme forms I and II were co-purified from a high-salt-Triton X-100 soluble extract of optic lobe by consecutive affinity chromatographies on procainamide- and concanavalin A-Sepharose columns and then separately obtained by preparative density gradient centrifugation. According to gel-filtration chromatography, sedimentation analysis and SDS-PAGE, the major form II is an amphiphilic globular dimer (135-136 kDa, 6.3-7.4 S) of monomers (66 kDa) S-S linked between terminal segments. Phosphatidylinositol anchors give cell membrane insertion, self-aggregation and detergent (Triton X-100, Brij 97) interaction. Form I, characterized only in part owing to its small amount, showed molecular size (129 kDa) and sedimentation coefficient (7.5 S) similar to those of form II; it is likely to be attached to the cell membrane by electrostatic interactions. Both forms behaved similarly with various inhibitors and underwent excess-substrate inhibition. The results obtained suggest a common origin of both form I and II from a single gene. The former could be a degradation product of the prevailing one (II), which is likely to be functional in cholinergic synapses.

Acute toxicity screening of novel AChE inhibitors using neuronal networks on microelectrode arrays.

Spontaneously active neuronal networks grown from embryonic murine frontal cortex on substrate integrated electrode arrays with 64 recording sites were used to assess acute neurobiological and toxic effects of a series of seven symmetrical, bifunctional alkylene-linked bis-thiocarbonate compounds designed to possess anticholinesterase activity. Acute functional neurotoxicity in the absence of cytotoxicity was defined as total collapse of spontaneous activity. All of the compounds were characterized as mixed inhibitors of AChE, with K(i)'s in the 10(-7)-10(-6) M range. The neuronal network assays revealed high repeatability for each compound, but surprisingly diverse effects among these closely related compounds. Six of the seven compounds produced changes in network activity at concentrations of 10-350 microM. Three of the compounds were excitatory, two were biphasic (excitatory at lower concentrations, inhibitory at higher), and one was solely inhibitory. Two of the inhibitory compounds produced irreversible inhibition of activity. Responses of cortical cultures to eserine were compared to the effects produced by the test compounds, with only one of seven providing a close match to the eserine profile. Matching of response patterns allows the classification of new drugs according to their response similarity to well-characterized agents. Spontaneously active neuronal networks reflect the interactions of multiple neurotransmitter and receptor systems, and can reveal unexpected side effects due to secondary binding. Utilizing such networks holds the promise of greater research efficiency through a more rapid recognition of physiological tissue responses.