Drug treatment in the development of mismatch repair defective acute leukemia and myelodysplastic syndrome

DNA from therapy-related acute leukemia/myelodysplastic syndrome cases (tAL/MDS) from the GIMEMA [Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto] Archive was examined for the microsatellite instability (MSI(+)) phenotype that is diagnostic for defective DNA mismatch repair. More than 60% (16/25) of tAL/MDS cases were MSI(+) in contrast to <4% (0/28) of de novo cases. hMLH1 gene silencing was rare and evidence of promoter methylation was found in less than one-third of the MSI(+) cases. Among the GIMEMA patients who had been treated for breast cancer there was an apparent trend towards early onset primary breast disease. This suggests that there might be common predisposing factors for breast cancer and tAL/MDS. There were also three examples of mutations in the MRE11 gene among the 25 tAL/MDS cases suggesting that defective recombinational DNA repair may promote the development of secondary malignancy. MSI(+) tAL/MDS was significantly associated with previous chemotherapy and the frequency of MSI(+) among radiotherapy patients was considerably lower. In view of the established relationship between drug resistance and mismatch repair defects, we suggest that selection for therapeutic drug resistance may contribute to the incidence of MSI(+) tAL/MDS.     

An easy‐to‐run method for routine analysis of eumelanin and pheomelanin in pigmented tissues

A procedure for analysis of melanin‐pigmented tissues based on alkaline hydrogen peroxide degradation coupled with high‐performance liquid chromatography (HPLC) ultraviolet determination of pyrrole‐2,3,5‐tricarboxylic acid (PTCA) for eumelanin and 6‐(2‐amino‐2‐carboxyethyl)‐2‐carboxy‐4‐hydroxybenzothiazole (BTCA) and 1,3‐thiazole‐2,4,5‐tricarboxylic acid for pheomelanin was recently developed. Despite advantages related to the degradation conditions and sample handling, a decrease of the reproducibility and resolution was observed after several chromatographic runs. We report herein an improved chromatographic methodology for simultaneous determination of PTCA and BTCA as representative markers of eumelanin and pheomelanin, respectively, based on the use of an octadecylsilane column with polar end‐capping with 1% formic acid (pH 2.8)/methanol as the eluant. The method requires conventional HPLC equipments and gives very good peak shapes and resolution, without need of ion pair reagents or high salt concentrations in the mobile phase. The intra‐assay precision of the analytical runs was satisfactory with CV values ≤4.0% (n = 5) for the two markers which did not exceed 8% after 50 consecutive injections on the column over 1 week. The peak area ratios at 254 and 280 nm (A₂₈₀/A₂₅₄: PTCA = 1.1, BTCA = 0.6) proved a valuable parameter for reliable identification of the structural markers even in the most complex degradation mixtures. The method can be applied to various eumelanin and pheomelanin pigmented tissues, including mammalian hair, skin and irides, and is amenable to be employed in population screening studies.     

Airborne fungi in an Italian rice mill

Summary Studies employing volumetric spore trap (VSP) and gravity settling culture plates (GSC) were conducted in order to analyse the air spora of a rice mill at Pavia, Italy, from October-December 1988. Results revealed a variety of fungal spores belonging to different genera and including recognized rice pathogenic fungi. The most frequent genera by GSC method includedAcremonium, Alternaria, Aspergillus, Aureobasidium, Cladosporium, Epicoccum, Fusarium, Helminthosporium, Mucor, Nigrospora, Penicillium, Rhizopus, Trichoderma, Trichothecium, and some unidentified fungi. Environmental assessment of fungal spores by VSP revealed that the most prevalent fungi were:Alternaria, Cladosporium, Epicoccum, Helminthosporium, Nigrospora, Pyricularia, Tilletia and hyaline, dark and coloured types of ascospores and basidiospores. Airborne fungal spore concentrations were particularly high (5,000–6,000 spores/m³) in the rooms of the rice mill where the initial stages of rough rice transformation take place, and dropped to 2,500 spores/m³ in the last room, where workers are. During a temporary interruption of the working processes, air spora concentration dropped below 1,000 spores/m³.Cladosporium, Epicoccum andNigrospora spores were predominant in all subdivisions of the indoor environments of the rice mill.     

Accumulation of copper and zinc from liquid manure in agricultural soils and crop plants

In Northern Italy it is a common practice to utilise slurries and other manure from intensive animal farming to fertilise agricultural soils. Due to the relatively high copper and zinc contents of these materials, this practice could lead to contamination of soils and the crops grown on them. In this study we have evaluated the extent of copper and zinc contamination of soils subjected to different levels of fertilisation with liquid manure and the copper and zinc concentrations in the edible tissues of three crops (maize, sugar beet and lucerne) grown on the same soils. There was a direct correlation between soil copper and zinc contents and the levels of application of animal wastes and copper concentrations were high enough to represent a risk, according to current European legislation. The metal contents in the edible tissues of the three crops were relatively low and variable, but showed no clear correlations with the intensity of liquid manure applications. Overall, there was no risk of contamination of the food chain, as all concentrations were well below levels considered to be toxic to animals.     

Distribution and Numbers of Pygmies in Central African Forests

Pygmy populations occupy a vast territory extending west-to-east along the central African belt from the Congo Basin to Lake Victoria. However, their numbers and actual distribution is not known precisely. Here, we undertake this task by using locational data and population sizes for an unprecedented number of known Pygmy camps and settlements (n = 654) in five of the nine countries where currently distributed. With these data we develop spatial distribution models based on the favourability function, which distinguish areas with favourable environmental conditions from those less suitable for Pygmy presence. Highly favourable areas were significantly explained by presence of tropical forests, and by lower human pressure variables. For documented Pygmy settlements, we use the relationship between observed population sizes and predicted favourability values to estimate the total Pygmy population throughout Central Africa. We estimate that around 920,000 Pygmies (over 60% in DRC) is possible within favourable forest areas in Central Africa. We argue that fragmentation of the existing Pygmy populations, alongside pressure from extractive industries and sometimes conflict with conservation areas, endanger their future. There is an urgent need to inform policies that can mitigate against future external threats to these indigenous peoples’ culture and lifestyles.     

The photochemical trapping rate from red spectral states in PSI-LHCI is determined by thermal activation of energy transfer to bulk chlorophylls

The average fluorescence decay lifetimes, due to reaction centre photochemical trapping, were calculated for wavelengths in the 690- to 770-nm interval from the published fluorescence decay-associated emission spectra for Photosystem I (PSI)-light-harvesting complex of Photosystem I (LHCI) [Biochemistry 39 (2000) 6341] at 280 and 170 K. For 280 K, the overall trapping time at 690 nm is 81 ps and increases with wavelength to reach 103 ps at 770 nm. For 170 K, the 690-nm value is 115 ps, increasing to 458 ps at 770 nm. This underlines the presence of kinetically limiting processes in the PSI antenna (diffusion limited). The explanation of these nonconstant values for the overall trapping time band is sought in terms of thermally activated transfer from the red absorbing states to the "bulk" acceptor chlorophyll (chl) states in the framework of the Arrhenius-Eyring theory. It is shown that the wavelength-dependent "activation energies" come out in the range between 1.35 and 2.7 kcal mol(-1), increasing with the emission wavelength within the interval 710-770 nm. These values are in good agreement with the Arrhenius activation energy determined for the steady-state fluorescence yield over the range 130-280 K for PSI-LHCI. We conclude that the variable trapping time in PSI-LHCI can be accounted for entirely by thermally activated transfer from the low-energy chl states to the bulk acceptor states and therefore that the position of the various red states in the PSI antenna seems not to be of significant importance. The analysis shows that the bulk antenna acceptor states are on the low-energy side of the bulk antenna absorption band.     

Basolateral Ca2+-dependent K+-channels play a key role in Cl- secretion induced by taurodeoxycholate from colon mucosa

The diarrhea associated with malabsorption of bile salts such as the secondary hydrophobic taurodeoxycholate (TDC) may be partly explained by the TDC-induced increase in colon Cl(-) secretion. We, therefore, investigated the effects of TDC (0.5-8 mM) on electrical parameters and electrolyte transport of rat proximal colon mucosa mounted in Ussing chambers. Colonic secretion, measured as short circuit current (I(SC)), progressively increased on mucosal incubation with TDC ranging 0.5-2 mM; up to TDC 2 mM, a spontaneous recovery toward control values with no changes in epithelial resistance (Rt), and lactate dehydrogenase (LDH) release was observed. In contrast, for TDC > 2 mM, I(SC) increased further and the effect was progressive and associated with a significant decrease in the Rt and increased LDH release, implying a cytolytic effect. Mucosal preincubation with the Cl(-) channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), fully prevented the precytolytic effect of TDC on I(SC). Serosal preincubation with furosemide, a Na(+)/K(+)/2Cl(-) cotransporter inhibitor, significantly reduced TDC-induced increase in I(SC). Inhibition of the basolateral Ca(2+)-dependent K(+) channel-rSK4-with serosal clotrimazole or incubation with mucosal Ca(2+)-free (EGTA) buffer completely prevented precytolytic TDC-induced increase in I(SC). In conclusion, Cl(-) secretion is activated in colon mucosa by TDC low concentrations; while at higher concentrations, a detergent cytotoxic effect intervenes. Activation of the Ca(2+)-dependent basolateral K(+) pathway, through TDC-induced apical Ca(2+) influx, provides the Na(+)/K(+)/2Cl(-) basolateral activation, thereby the driving force for the apical exit of Cl(-) ions. These findings further enhance the knowledge of the pathogenic mechanisms of diarrhea associated with bile salt malabsorption.     

The common Scandinavian human leucocyte antigen ancestral haplotype 62.1 as prognostic factor in patients with advanced malignant melanoma

Purpose  We have previously demonstrated an association of the human leukocyte antigen (HLA), HLA-A2 allele with ovarian and prostate cancer mortality as well as a segregation of the ancestral HLA haplotype (AHH) 62.1 [(A2) B15 Cw3 DRB1*04] in patients with stage III–IV serous ovarian cancer. The objective of the present study was to determine the role of the HLA phenotype on the prognosis in stage III–IV malignant melanoma patients. Patients and methods  A cohort of metastatic malignant melanoma patients (n = 91), in stage III (n = 26) or IV (n = 65) were analysed for HLA-A, -B, -Cw and -DRB1 types by PCR/sequence-specific primer method. The frequencies of HLA alleles in the patients were compared to that of healthy Swedish bone marrow donors. The effect of HLA types on prognosis was defined by Kaplan–Meier and Cox analysis. Results  The presence of the AHH 62.1 in clinical stage IV patients was significantly and independently associated with the worst survival rate recorded from the appearance of metastasis (HR = 2.14; CI = 1.02–4.4; P = 0.04). In contrast, the period from the primary diagnosis to metastasis was the longest in patients with this haplotype (HR = 0.40; CI = 0.17–0.90; P = 0.02). Conclusions  Melanoma patients in our cohort with 62.1 AHH which is associated with autoimmune diseases have an initial strong anti-tumour control with longer metastasis-free period. These patients have rapid progression after the appearance of metastasis, responding poorly to chemo- or/and immunotherapy. This apparently paradoxical clinical process could be due to the interplay between tumour clones escape and immune surveillance ending up with a rapid disease progression. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Structure of a novel class II phospholipase D: catalytic cleft is modified by a disulphide bridge

Phospholipases D (PLDs) are principally responsible for the local and systemic effects of Loxosceles envenomation including dermonecrosis and hemolysis. Despite their clinical relevance in loxoscelism, to date, only the SMase I from Loxosceles laeta, a class I member, has been structurally characterized. The crystal structure of a class II member from Loxosceles intermedia venom has been determined at 1.7 Å resolution. Structural comparison to the class I member showed that the presence of an additional disulphide bridge which links the catalytic loop to the flexible loop significantly changes the volume and shape of the catalytic cleft. An examination of the crystal structures of PLD homologues in the presence of low molecular weight compounds at their active sites suggests the existence of a ligand-dependent rotamer conformation of the highly conserved residue Trp230 (equivalent to Trp192 in the glycerophosphodiester phosphodiesterase from Thermus thermophofilus, PDB code: 1VD6) indicating its role in substrate binding in both enzymes. Sequence and structural analyses suggest that the reduced sphingomyelinase activity observed in some class IIb PLDs is probably due to point mutations which lead to a different substrate preference.