Determination of organophosphorus pesticide residues in human tissues by capillary gas chromatography-negative chemical ionization mass spectrometry analysis

We describe an analytical method that allows the determination of organophosphorus pesticides (OPs) in different human tissues. It involves an extraction procedure with ethanol-ethyl acetate, followed by gel permeation chromatography clean-up step and analysis by capillary gas chromatography-negative chemical ionization mass spectrometry in the selected ion monitoring mode. The method was tested for 37 OPs and the recoveries obtained vary between 60 and 106% with standard deviations ranging between +/-2 and +/-10. These values are independent of the analyzed tissue. Peak area repeatability as RSD for some OPs was < or =4.8% while a good linear relationship in the range 1.0-500 pg microl(-1) with r(2)> or =0.9878 was obtained. The limit of detection for the 37 OPs falls between 0.01 and 0.09 ng g(-1) with an RSD< or =9.5%. The analytical set up in this paper has been used to analyze different samples of human tissues (liver, healthy kidney, cancer kidney and adipose tissue) of 24 patients. The number of the identified OPs in the tissue samples is different (max. 20) according to the sample while their concentration ranges between the limit of detection and 28.0 ng g(-1). The highest concentrations have been determined in liver samples without any pathology (0.4-28.0 ng g(-1)) while the lowest concentrations have been determined in healthy kidney samples (0.01-1.50 ng g(-1)). In the cancer kidney samples OP concentrations vary between 0.03 and 4.6 ng g(-1): these concentrations are more elevated than those determined in healthy kidney samples. The comparison between the concentration of OPs determined in the healthy part, when possible, and those determined in the cancer part of the same kidney sample are very interesting: in fact, in the latter the OP concentration is generally 1-2-times higher than that in the former, an index of lower enzymatic activity in the cancer tissue.     

Apoptosis: Molecular regulation of cell death and hematologic malignancies

We describe the molecular mechanisms of apoptosis and its relationships with hematologic malignancies, stressing the concept that, both positive and negative deregulation of apoptosis, may be involved in hematologic human diseases. So, this fundamental process must be balanced by so far unknown mechanisms, involving caspases (cysteine proteases, cleaving the protein substrate after an aspartate residue). These, so far known, ten proteases, are interconnected in a molecular cascade, initiated by the release of cytochrome C from mitochondrial membranes and its interaction with APAF-1 (the homolog of the Caenorhabditis e. CED-4) and with caspase 9, that initiates the proteolitic cascade (1,2). The conclusion is that apoptosis is a very important process, but yet poorly known in molecular details, in spite of the efforts of many scientists. Even the role of bcl-2, the main gene protecting from apoptosis, is still unknown. We close this chapter with a list of ten different technical approaches that can be useful tools to study apoptosis, and tracing the molecular principles on which they are based.     

Analysis of 22 deletion breakpoints in dystrophin intron 49

Over 60% of Duchenne and Becker muscular dystrophies are caused by deletions spanning tens or hundreds of kilobases in the dystrophin gene. The molecular mechanisms underlying the loss of DNA at this genomic locus are not yet understood. By studying the distribution of deletion breakpoints at the genomic level, we have previously shown that intron 49 exhibits a higher relative density of breakpoints than most dystrophin introns. To determine whether the mechanisms leading to deletions in this intron preferentially involve specific sequence elements, we sublocalized 22 deletion endpoints along its length by a polymerase-chain-reaction-based approach and, in particular, analyzed the nucleotide sequences of five deletion junctions. Deletion breakpoints were homogeneously distributed throughout the intron length, and no extensive homology was observed between the sequences adjacent to each breakpoint. However, a short sequence able to curve the DNA molecule was found at or near three breakpoint junctions.     

Probing the hirudin-thrombin interaction by incorporation of noncoded amino acids and molecular dynamics simulation

Thrombin is a primary target for the development of novel anticoagulants, since it plays two important and opposite roles in hemostasis: procoagulant and anticoagulant. All thrombin functions are influenced by Na+ binding, which triggers the transition of this enzyme from an anticoagulant (slow) form to a procoagulant (fast) form. In previous studies, we have conveniently produced by chemical synthesis analogues of the N-terminal fragment 1-47 of hirudin HM2 containing noncoded amino acids and displaying up to approximately 2700-fold more potent antithrombin activity, comparable to that of full-length hirudin. In the work presented here, we have exploited the versatility of chemical synthesis to probe the structural and energetic properties of the S3 site of thrombin through perturbations introduced in the structure of hirudin fragment 1-47. In particular, we have investigated the effects of systematic replacement of Tyr3 with noncoded amino acids retaining the aromatic nucleus of Tyr, as well as similar hydrophobic and steric properties, but possessing different electronic (e.g., p-fluoro-, p-iodo-, or p-nitro-Phe), charge (p-aminomethyl-Phe), or conformational (homo-Phe) properties. Our results indicate that the affinity of fragment 1-47 for thrombin is proportional to the desolvation free energy change upon complex formation, and is inversely related to the electric dipole moment of the amino acid side chain at position 3 of hirudin. In this study, we have also identified the key features that are responsible for the preferential binding of hirudin to the procoagulant (fast) form of thrombin. Strikingly, shaving at position 3, by Tyr --> Ala exchange, abolishes the differences in the affinity for thrombin allosteric forms, whereas a bulkier side chain (e.g., beta-naphthylalanine) improves binding preferentially to the fast form. These results provide strong, albeit indirect, evidence that the procoagulant (fast) form of thrombin is in a more open and accessible conformation with respect to the less forgiving structure it acquires in the slow form. This view is also supported by the results of molecular dynamics simulations conducted for 18 ns on free thrombin in full explicit water, showing that after approximately 5 ns thrombin undergoes a significant conformational transition, from a more open conformation (which we propose can be related to the fast form) to a more compact and closed one (which we propose can be related to the slow form). This transition mainly involves the Trp148 and Trp60D loop, the S3 site, and the fibrinogen binding site, whereas the S1 site, the Na+-binding site, and the catalytic pocket remain essentially unchanged. In particular, our data indicate that the S3 site of the enzyme is less accessible to water in the putative slow form. This structural picture provides a reasonable molecular explanation for the fact that physiological substrates related to the procoagulant activity of thrombin (fibrinogen, thrombin receptor 1, and factor XIII) orient a bulky side chain into the S3 site of the enzyme. Taken together, our results can have important implications for the design of novel thrombin inhibitors, of practical utility in the treatment of coagulative disorders.     

Evaluation of the surface-averaged load exerted on a blood element by the Reynolds shear stress field provided by artificial cardiovascular devices

Implantable prosthetic devices can often affect the recipient's hemostasis, with possible hemolysis and thrombus formation. Since such devices can produce turbulent flow, it is important to characterize it as accurately as possible, by means of the Reynolds stress tensor. Some parameters related to the latter have been often used to provide a quantity related to the possible damage to blood constituents: the TSS_max, for instance, has been associated with hemolysis. It can be expressed as TSS_max=(σ₁−σ₃)/2, σ₁ and σ₃ being the highest and lowest principal normal stresses (PNSs) in each point of the flow.

In the present work, the average value of the shear stress over a spherical surface, representative of a blood component, is derived. All three PNSs (σ₁, σ₂ and σ₃) are found to have an equal role in the determination of this parameter, since the relative formula shows a marked symmetry with respect to the PNSs. The average shear stress level, for a given (σ₁, σ₃) pair (hence, for a given TSS_max), has a minimum and maximum value, depending on the particular σ₂ value yielded by the local structure of the turbulent flow field. A numerical investigation on more complex geometries shows similar results. The role of the intermediate PNS is thus shown for the first time to have a physical relevance. The presented results can be useful whenever a spatial averaging of the shear field is important to be assessed, such as in the case of platelet activation. A new parameter is thus proposed, which can be correlated with prosthetic devices complications.     

Estrogens and male reproduction

The role of estrogen on male reproductive function has become clearer in the last decade. During these years the study of the effect of testosterone, estrogen or an aromatase inhibitor in hypogonadal men provided a first evidence of the effects of estrogens in the regulation of gonadotropin secretion. At the same time, the development of a line of transgenic male mice lacking estrogen receptor α, estrogen receptor β or aromatase gene provided further evidence about the role of estrogens not only in the regulation of gonadotropin secretion, but also on the effects of estrogens on testicular function and development. A confirmation of these actions of estrogens came from the observation of naturally occurring mutations of the estrogen receptor and of the aromatase gene in human males. Based on these data it has been demonstrated that estrogens are major regulators of gonadotropin secretion acting both at pituitary and hypotalamic level. The presence in the human reproductive structures of estrogen receptor α, estrogen receptor β and the aromatase enzyme indicates the existence of receptor α, estrogen receptor β or aromatase estrogen actions at this level. Anyway, the precise role of estrogens in testicular development and function and on the regulation of human spermatogenesis has not yet been precisely clarified.     

Carcinogenesis in laboratory mice after low doses of ionizing radiation

Experimental data on the incidence of solid tumors from various long-term mouse studies performed at the Casaccia laboratories over several years were reconsidered, limiting the analysis to the results available for doses equal to or less than 17 cGy of neutrons and 32 cGy of X rays since these dose limits are reasonably close to the generally accepted low-dose levels for high- and low-LET radiation (i.e. D(high-LET) < 5 cGy and D(low-LET) < 20 cGy, respectively). The following long-term experiments with BC3F1 mice were reviewed: (a) females treated with single doses of 1.5 MeV neutrons or 250 kVp X rays, (b) males treated with fractionated doses of fission neutrons, and (c) mice of both sexes irradiated in utero 17.5 days post coitus with single doses of fission neutrons or X rays. An experiment with CBA mice of both sexes treated with single doses of fission neutrons was also included in this study. Analysis was done on animals at risk; thus all incidences of tumor-bearing animals were expressed as the percentage excess incidence with respect to the controls. Ovarian tumors and other solid neoplasms were considered. The percentage frequencies and mean survival times of tumor-free mice were also recalculated. The results indicate the existence of a region at low doses where the final incidence of solid neoplasms is indistinguishable from the background incidence. These data reinforce the idea that at low doses the effectiveness of ionizing radiation in inducing solid neoplasms in laboratory mice is very low.