Study of molecular mechanisms of pro-apoptotic activity of NCX 4040, a novel nitric oxide-releasing aspirin, in colon cancer cell lines

Background  

Despite numerous studies aimed at verifying the antitumor activity of nitric oxide-releasing nonsteroidal antiflammatory drugs (NO-NSAIDs), little is known about the molecular targets responsible for their antineoplastic properties. In the present study, we investigated the mechanisms underlying the cytotoxicity of NCX 4040, a novel NO-aspirin with promising antineoplastic action, in in vitro human colon cancer models.     

A novel method for rapid genotypic identification of alpha 1-antitrypsin variants.

There is worldwide growing awareness of alpha 1-antitrypsin deficiency (AATD), a major hereditary disorder in Caucasians. The gold standard for laboratory diagnosis of AATD is thin-layer isoelectrofocusing (IEF), which is labor intensive and should be performed in reference laboratories. The aim of this study was to find an easy, fast, and cheap method for detecting alpha1-antitrypsin S and Z variants, the most frequent variants associated with AATD. The novel method herein described is based on SexAI/Hpy99I RFLP. We studied samples from 90 subjects enrolled in the Italian National Registry for AATD, previously typed by isoelectrofocusing. We found a complete agreement among our results, IEF, and genotypes obtained by standard methods. We concluded that this novel method combines efficiency, ease, swiftness, and low cost.     

Hypomethylation of the human HLA-DRα gene in breast carcinomas and autologous metastases

The methylation pattern of the human HLA-DR gene was analyzed in normal breast tissues, breast primary tumors and lymphonodal metastases isolated from patients carrying breast carcinomas. In breast adenomas and also in normal tissues (including breast, muscle, brain, sperm and T- and B-lymphocytes), the HLA-DR gene is hypermethylated at the CCGG and GCGC sites. In all tissues studied, the only constantly unmethylated region is located in the 5 portion of the gene, near the promoter sequence. Further, the results indicate that the HLA-DR gene is hypomethylated in carcinomas and in the relative metastatic lymph nodes. It is suggested that hypomethylation of the human HLA-DR gene could be proposed as a molecular marker of malignant breast tumors.     

Effects of prolonged cycle ergometer exercise on maximal muscle power and oxygen uptake in humans

Summary The mechanical power (W_tot, W·kg^–1) developed during ten revolutions of all-out periods of cycle ergometer exercise (4–9 s) was measured every 5–6 min in six subjects from rest or from a baseline of constant aerobic exercise [50%–80% of maximal oxygen uptake (VO_2max)] of 20–40 min duration. The oxygen uptake [VO₂ (W·kg^–1, 1 ml O₂ = 20.9 J)] and venous blood lactate concentration ([la]_b, mM) were also measured every 15 s and 2 min, respectively. During the first all-out period, W_tot decreased linearly with the intensity of the priming exercise (W_tot = 11.9–0.25·VO₂). After the first all-out period (i greater than 5–6 min), and if the exercise intensity was less than 60% VO_2max, W_tot, VO₂ and [la]_b remained constant until the end of the exercise. For exercise intensities greater than 60% VO_2max, VO₂ and [la]_b showed continuous upward drifts and W_tot continued decreasing. Under these conditions, the rate of decrease of W_tot was linearly related to the rate of increase of V [(d W_tot/dt) (W·kg^–1·s^–1) = 5.0·10^–5 –0.20·(d VO₂/dt) (W·kg^–1·s^–1)] and this was linearly related to the rate of increase of [la]_b [(d VO₂/dt) (W·kg^–1·s^–1) = 2.310^–4 + 5.910^–5·(d [la]_b/dt) (mM·s^–1)]. These findings would suggest that the decrease of W_tot during the first all-out period was due to the decay of phosphocreatine concentration in the exercising muscles occurring at the onset of exercise and the slow drifts of VO₂ (upwards) and of W_tot (downwards) during intense exercise at constant W_tot could be attributed to the continuous accumulation of lactate in the blood (and in the working muscles).     

Absence of TREM2 polymorphisms in patients with Alzheimer's disease and Frontotemporal Lobar Degeneration

Triggering Receptor Expressed on Myeloid cells (TREM)2 deficiency originates a genetic syndrome characterized by bone cysts and presenile dementia, named Nasu-Hakola disease (NHD). Early onset dementia and marked involvement of frontal regions are features characterizing both NHD and other kinds of neurodegenerative disorders, such as Frontotemporal Lobar Degeneration (FTLD), and, in some cases, Alzheimer's disease (AD). Three Single Nucleotide Polymorphisms (SNPs) in TREM2 coding region were screened by allelic discrimination in a population of probable AD patients as well as FTLD patients as compared with age-matched controls. In addition, mutation scanning of the coding region of TREM2 gene was carried out in 7 patients with early onset AD (EOAD), 16 FTLD, and 20 controls. None of the SNPs analyzed was present, either in patients or controls. Moreover, mutation scanning of the five exons of TREM2 failed to detect the presence of novel polymorphisms. These data demonstrate that TREM2 coding region is highly conserved, implying a crucial role of this receptor. Further studies, including a functional analysis, are certainly required to clarify the role of TREM2 in neurodegenerative processes.     

Long distance transport of ragweed pollen as a potential cause of allergy in central Italy.

BACKGROUND: Ambrosia pollen is an important allergen in North America and, as recently discovered, in some European countries. In Italy, the most affected area is the northeast, whereas ragweed has not been reported in the central and southern parts of the country. OBJECTIVE: To identify the source of ragweed pollen detected in Florence and Pistoia in central Italy. METHODS: Ragweed pollen data were collected in Florence and Pistoia for a 6-year period (1999-2004). The relationship between pollen counts and local ground prevalent wind directions was evaluated with analysis of variance and the least significant difference test. Weather conditions were also evaluated on a large-scale circulation pattern by analyzing weather maps and air mass back trajectories. RESULTS: A highly statistically significant relationship between daily prevailing wind direction and pollen count was found in the period under investigation; the ragweed pollen peaks were recorded when winds from northeast in Florence and north-northeast in Pistoia were observed. The synoptic weather situation and the path of back trajectories suggest an area around southern Hungary as a possible source of Ambrosia pollen. Furthermore, the pollen count was above the clinical threshold several times in both Florence and Pistoia. CONCLUSIONS: Several factors indicate that the detection of ragweed pollen in central Italy is due to long distance transport. Taking into consideration the high allergenicity of Ambrosia pollen, the present findings, if confirmed, suggest that the number of sensitized individuals might significantly increase in the near future.     

Myosin light chain kinase modulates hypotonicity-induced Ca2+ entry and Cl– channel activity in human cervical cancer cells

Hypotonicity-induced Ca2+ entry is a critical signal for the normal regulatory volume decrease in human cervical cancer cells. The aim of this study was to explore the role of myosin light chain kinase (MLCK) in the regulation of hypotonicity-induced Ca2+ signalling and Cl- channel activity. Blockade of MLCK activity by MLCK(11-19) amide, a substrate-specific peptide inhibitor, markedly attenuated hypotonicity-induced Ca2+ entry. A similar result was obtained with ML-7, a synthetic naphthalenesulphonyl derivative that inhibits the binding of ATP to MLCK. More than 85% of the activity of the volume-regulated Cl- channel was suppressed when intracellular Ca2+ was buffered to near zero in the absence of extracellular Ca2+, suggesting that hypotonicity-induced Ca2+ signalling is important for the activation of the volume-regulated Cl- channel. Intracellular dialysis with MLCK(11-19) amide or ML-7 concentration-dependently reduced the amplitude and rate of activation of the volume-regulated Cl- channel. Swelling-activated taurine transport was also inhibited concentration dependently by ML-7 and MLCK(11-19) amide with IC(50) values of 6.4 and 2.0 microM, respectively. Hypotonicity induced MLC phosphorylation which was mediated totally by MLCK and depended on Ca2+ entry. However, phosphorylated MLC per se was not involved critically in the regulation of Ca2+ entry and activation of volume-sensitive organic osmolyte/anion channels (VSOAC). We propose that MLCK has a novel function in regulating the activation of VSOAC by mediating Ca2+ entry in response to hypotonicity. This function of MLCK on Ca2+ signalling does not correlate with MLC phosphorylation.     

Agrobacterium-mediated DNA transfer

Summary Agrobacterium tumefaciens naturally transfers DNA into plant cells and is clearly one of the most effective methods of directed DNA transfer presently available. Two kinds of vectors are commonly used. Cointegrative vectors have the foreign genes incorporated directly into the Ti plasmid. Binary vectors carry two plasmids; the main Ti plasmid where most of the T-DNA has been removed, and a second plasmid containing the foreign genes between the usual border sequences. The vir genes on the main plasmid function to mobilize the foreign genes into a plant cell. Most plant transformation methods follow the procedure of cocultivating wounded tissue with vir-gene-induced bacteria. The cocultivation step is followed by transfer to a selective medium containing antibiotics to kill the bacterium and to allow only growth of transformed tissue. Several selectable markers are available that include resistance to antibiotics, herbicides, or drugs. In addition, several scorable markers such as the bacterial glucuronidase, chloramphenicol acetyl transferase, and the Agrobacterium opine genes are used to verify transformation. Southern blotting and inheritance of transferred genes are ultimately used to demonstrate stable transformation.     

Recovery of the endogenous beta cell function in the NOD model of autoimmune diabetes

In light of accumulating evidence that the endocrine pancreas has regenerative properties and that hematopoietic chimerism can abrogate destruction of beta cells in autoimmune diabetes, we addressed the question of whether recovery of physiologically adequate endogenous insulin regulation could be achieved in the nonobese diabetic (NOD) mice rendered allogeneic chimerae. Allogeneic bone marrow (BM) was transplanted into NOD mice at the preclinical and overtly clinical stages of the disease using lethal and nonlethal doses of radiation for recipient conditioning. Islets of Langerhans, syngeneic to the BM donors, were transplanted under kidney capsules of the overtly diabetic animals to sustain euglycemia for the time span required for recovery of the endogenous pancreas. Nephrectomies of the graft-bearing organs were performed 14 weeks later to confirm the restoration of endogenous insulin regulation. Reparative processes in the pancreata were assessed histologically and immunohistochemically. The level of chimerism in NOD recipients was evaluated by flow cytometric analysis. We have shown that as low as 1% of initial allogeneic chimerism can reverse the diabetogenic processes in islets of Langerhans in prediabetic NOD mice, and that restoration of endogenous beta cell function to physiologically sufficient levels is achievable even if the allogeneic BM transplantation is performed after the clinical onset of diabetes. If the same pattern of islet regeneration were shown in humans, induction of an autoimmunity-free status by establishment of a low level of chimerism, or other alternative means, might become a new therapy for type 1 diabetes.