Neuroendocrine aspects of migraine in women

Migraine is a complex disabling disease influenced mainly by age and gender during the life span. Neuroendocrine events related to reproductive stages and to the menstrual cycle may cause significant change in the clinical pattern of migraine over time, as a consequence of failure in adaptation higher in women than in men. Indeed, the individual threshold of vulnerability to manifest migraine is modulated by hormonal fluctuations naturally occurring throughout the menstrual cycle and at the time of reproductive transitions. In the present short review, the role of endogenous estrogen at the level of brain circuitries which are involved in multiple cellular, neurochemical and neurophysiological processes associated with migraine will be summarized in the context of reproductive milestones. In addition, some clues to recognize hormonally sensitive women on the basis of their migraine history, i.e. onset, association with menstruation or premenstrual syndrome, course during pregnancy and menopause, will be discussed in order to expand the knowledge of reproductive endocrinology in the management of migraine in women.     

KH domains with impaired nucleic acid binding as a tool for functional analysis

In eukaryotes, RNA-binding proteins that contain multiple K homology (KH) domains play a key role in coordinating the different steps of RNA synthesis, metabolism and localization. Understanding how the different KH modules participate in the recognition of the RNA targets is necessary to dissect the way these proteins operate. We have designed a KH mutant with impaired RNA-binding capability for general use in exploring the role of individual KH domains in the combinatorial functional recognition of RNA targets. A double mutation in the hallmark GxxG loop (GxxG-to-GDDG) impairs nucleic acid binding without compromising the stability of the domain. We analysed the impact of the GDDG mutations in individual KH domains on the functional properties of KSRP as a prototype of multiple KH domain-containing proteins. We show how the GDDG mutant can be used to directly link biophysical information on the sequence specificity of the different KH domains of KSRP and their role in mRNA recognition and decay. This work defines a general molecular biology tool for the investigation of the function of individual KH domains in nucleic acid binding proteins.     

Neuroprotective effect of cannabidiol, a non-psychoactive component from Cannabis sativa, on beta-amyloid-induced toxicity in PC12 cells

Abstract Alzheimer's disease is widely held to be associated with oxidative stress due, in part, to the membrane action of beta-amyloid peptide aggregates. Here, we studied the effect of cannabidiol, a major non-psychoactive component of the marijuana plant (Cannabis sativa) on beta-amyloid peptide-induced toxicity in cultured rat pheocromocytoma PC12 cells. Following exposure of cells to beta-amyloid peptide (1 micro g/mL), a marked reduction in cell survival was observed. This effect was associated with increased reactive oxygen species (ROS) production and lipid peroxidation, as well as caspase 3 (a key enzyme in the apoptosis cell-signalling cascade) appearance, DNA fragmentation and increased intracellular calcium. Treatment of the cells with cannabidiol (10(-7)-10(-4)m) prior to beta-amyloid peptide exposure significantly elevated cell survival while it decreased ROS production, lipid peroxidation, caspase 3 levels, DNA fragmentation and intracellular calcium. Our results indicate that cannabidiol exerts a combination of neuroprotective, anti-oxidative and anti-apoptotic effects against beta-amyloid peptide toxicity, and that inhibition of caspase 3 appearance from its inactive precursor, pro-caspase 3, by cannabidiol is involved in the signalling pathway for this neuroprotection.     

High-fat diet alters the oligosaccharide chains of colon mucins in mice

Mucins are high molecular weight epithelial proteins, strongly glycosylated, and are the main component of the mucus. Since mucus secretion can be altered in diseases, colon mucins can be regarded as a biomarker of chronic inflammatory bowel diseases or preneoplastic changes. Conventional histochemistry and lectin histochemistry combined with chemical treatment and enzymatic digestion were carried out to analyze the colon mucins in mice fed a high-fat diet for 25 weeks, a period sufficient to induce simple liver steatosis, to check whether the carbohydrate features of mucus can be altered by an inadequate diet. An increase in the sialo/sulfomucins ratio with respect to control mice, assessed by computerized image analysis, was observed in the colon, although differences in sialic acid acetylation between control and mice fed a high-fat diet were not found. High-fat diet was also associated with altered lectin-binding pattern of the mucus, with a probable shortening of oligosaccharide chains of glycoproteins. This pattern was leading to over-expression of Galβ1,3GalNAc terminal dimers (TF antigen) and GalNAc terminal residues (Tn antigen). This altered composition of mucins can be related to a defect in the process of glycosylation, or to incomplete maturation of goblet cells, and may be an early indication of preneoplastic and neoplastic changes. In conclusion, our findings confirm that a fatty-rich diet (Western-style diet) induces alteration of mucins and may be associated with colon diseases. Our investigation corroborates the usefulness of lectins histochemistry in the early diagnosis of prepathological states of the colon.     

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.     

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.     

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.     

Cystatin B and its EPM1 mutants are polymeric and aggregate prone in vivo

Progressive myoclonus epilepsy type 1 (EPM1) is a neurodegenerative disease correlating with mutations of the cystatin B gene. Cystatin B is described as a monomeric protein with antiprotease function. This work shows that, in vivo, cystatin B has a polymeric structure, highly resistant to SDS, urea, boiling and sensitive to reducing agents and alkaline pH. Hydrogen peroxide increases the polymeric structure of the protein. Mass spectrometry analysis shows that the only component of the polymers is cystatin B. EPM1 mutants of cystatin B transfected in cultured cells are also polymeric. The banding pattern generated by a cysteine-minus mutant is different from that of the wild-type protein as it contains only monomers, dimers and some very high MW bands while misses components of MW intermediate between 25 and 250 kDa. Overexpression of wild-type or EPM1 mutants of cystatin B in neuroblastoma cells generates cytoplasmic aggregates. The cysteine-minus mutant is less prone to the formation of inclusion bodies. We conclude that cystatin B in vivo has a polymeric structure sensitive to the redox environment and that overexpression of the protein generates aggregates. This work describes a protein with a physiological role characterized by highly stable polymers prone to aggregate formation in vivo.