Nucleophosmin mutations alter its nucleolar localization by impairing G-quadruplex binding at ribosomal DNA

Nucleophosmin (NPM1) is an abundant nucleolar protein implicated in ribosome maturation and export, centrosome duplication and response to stress stimuli. NPM1 is the most frequently mutated gene in acute myeloid leukemia. Mutations at the C-terminal domain led to variant proteins that aberrantly and stably translocate to the cytoplasm. We have previously shown that NPM1 C-terminal domain binds with high affinity G-quadruplex DNA. Here, we investigate the structural determinants of NPM1 nucleolar localization. We show that NPM1 interacts with several G-quadruplex regions found in ribosomal DNA, both in vitro and in vivo. Furthermore, the most common leukemic NPM1 variant completely loses this activity. This is the consequence of G-quadruplex–binding domain destabilization, as mutations aimed at refolding the leukemic variant also result in rescuing the G-quadruplex–binding activity and nucleolar localization. Finally, we show that treatment of cells with a G-quadruplex selective ligand results in wild-type NPM1 dislocation from nucleoli into nucleoplasm. In conclusion, this work establishes a direct correlation between NPM1 G-quadruplex binding at rDNA and its nucleolar localization, which is impaired in the acute myeloid leukemia-associated protein variants.     

Phage display for the production of human monoclonal antibodies against human pathogens

In the last decade an increasing number of antibodies have made their way from the research benchtops into the clinics and many more are currently under clinical trial. Among monoclonal antibody-producing techniques, phage-display is undoubtedly the most effective and versatile. Cloning of the entire humoral repertoire derived from an infected patients into a phage display vector allows not only the simple generation of monoclonal antibodies of desired specificity, but also the molecular dissection of the antibody response itself. Generation of large panels of human monoclonal antibodies against human pathogens could open new perspectives in understanding the interplay between the infectious agent and the infected host providing tools for the prevention and the therapy of human communicable diseases. In this paper the basic principles of the phage-display approach as well as its most recent applications are reviewed.     

Prevalence of human herpesvirus (HHV)-8 infection among carriers of cardiovascular disease

Infectious agents, such as herpesviruses, have been hypothesized to be involved in development of atheromatous plaque. The study aim was to evaluate the possibility that HHV-8 infection could be an additional risk factor for the establishment of cardiovascular disease. HHV-8 seroprevalence was determined by immunofluorescence in a population of cardiovascular disease patients (n=50) as compared to an age- and sex-matched group of control subjects (n=47); HHV-8 genome was detected in DNA extracted from circulating PBMC and from atheromatous lesions by PCR with primers specific for the minor virus capsid gene (ORF 26). The seroprevalence of HHV-8 was significantly increased in the patients as compared to the control population, while the presence of HHV-8 genome was observed in PBMC from 2 patients and 1 control. Virus-specific DNA was found in 2 out of 4 atheromatous plaques. The higher seroprevalence in patients suffering from vascular diseases as compared to age-and sex-matched controls suggests that HHV-8 infection could be an additional risk factor for the establishment of cardiovascular disease, although the data on the persistence of viral DNA in PBMC or in the arterial lesions are too exiguous to definitively support this hypothesis. More extensive studies are needed to define the exact role of HHV-8 infection in the establishment and progression of atheromatous lesions.     

Mean-square stability of a stochastic model for bacteriophage infection with time delays

We consider the stability properties of the positive equilibrium of a stochastic model for bacteriophage infection with discrete time delay. Conditions for mean-square stability of the trivial solution of the linearized system around the equilibrium are given by the construction of suitable Lyapunov functionals. The numerical simulations of the strong solutions of the arising stochastic delay differential system suggest that, even for the original non-linear model, the longer the incubation time the more the phage and bacteria populations can coexist on a stable equilibrium in a noisy environment for very long time.     

Lack of an association between candidate gene loci and idiopathic generalized epilepsy in Kuwaiti Arab children

Summary Idiopathic generalized epilepsies (IGEs) are the most common types of epilepsy in childhood and adolescence. A variety of data suggest that IGEs have a predominant genetic etiology. Recently, a number of gene mutations have been found to be associated with various types of epilepsy in mainly the Caucasian populations. The objective of this study was to investigate the association of three different candidate genes with IGE in Kuwaiti Arab children. This study includes 123 Kuwaiti patients with a confirmed diagnosis of epilepsy. Most of the patients have had a diagnostic EEG with generalized spike-wave discharges (GSWs). All patients were evaluated by using a validated seizure questionnaire. The clinical type of epilepsy was determined by a trained neurologist/pediatrician. The study also include 100 controls, the control subjects were children which did not have any history of neurological disorders. Blood samples were collected from all patients and control subjects after taking informed consent. DNA was isolated and analyzed by molecular methods. A FokI polymorphism in neuronal nicotinic acetylcholine receptor alpha-4 subunit (CHRNA4) gene was detected by PCR-RFLP method. A missense mutation (Ser248Phe) in CHRNA4 gene was analyzed by PCR-RFLP using HpaII. A C121W mutation in sodium-channel beta-1 subunit (SCN1B) gene was screened by a PCR-RFLP method using HinPI. A 2-bp deletion in Cystatin B gene was detected by PCR-RFLP using XcmI. The incidence of three FokI polymorphism genotypes in Kuwaiti IGE patients was 1,1 (85%), 1,2 (14%) and 2,2 (1%) respectively. The missense mutation Ser248Phe of CHRNA4 gene was not detected at all in Kuwaiti IGE patients. The C387G transversion resulting in C121W change in third exon of the SCN1B gene was detected in 3/123 patients (2%). The patients carrying this mutation also exhibited febrile seizures. The incidence of 2 bp deletion in the cystatin B gene was found to be 4% (5/123 IGE patients). The data obtained from molecular analysis show a lack of association between three candidate genes and clinical expression of IGE in Kuwaiti Arab children. This is completely different from the findings reported from Caucasian populations of France, Australia and USA in which case a strong association has been reported between IGE and these genes. To whom corresspondence should be addressed. Tel: +965-5319486; Fax: +965-5338940; E-mail: haider@hsc.edu.kw     

Engraftment and differentiation of neocortical progenitor cells transplanted to the embryonic brain in utero

Transplantation of neural progenitors or stem cells is a most useful tool to investigate the relative contribution of cell-autonomous mechanisms and environmental cues in the regulation of cell specification and differentiation during CNS development. To assess the capability of neocortical progenitor cells to integrate into foreign brain regions, here we examined the fate of precursor cells isolated from the dorsal telencephalon of E12 ß-actin-EGFP transgenic mouse embryos after heterotopic/heterochronic transplantation to the E16 rat brain in utero. Our observations show that donor cells were able to penetrate, survive and produce mature cell types into wide regions of the host CNS. Namely, EGFP-positive cells acquired site-specific neuronal identities in many telencephalic regions, including neocortex, hippocampus, olfactory bulb and corpus striatum. In contrast, incorporation into more caudal sites was much less efficient. A fraction of donor cells formed large aggregates that remained segregated from the host milieu. Such aggregates contained mature neurons and glia, including some EGFP-negative elements of host origin, and developed the complex organization of the mature nervous tissue. On the other hand, transplanted cells that engrafted in the parenchyma of extratelencephalic regions predominantly generated glial types. The few neurons failed to acquire obvious site-specific phenotypic traits and did not integrate into the local host architecture. Altogether, our observations indicate that E12 neocortical progenitors are already committed towards regional identities and are unable to modify their phenotypic choices when exposed to heterotopic environmental conditions along different rostro-caudal domains of the embryonic CNS.     

Biostimulant activity of humic substances extracted from leonardites

Background and aims

Biostimulants are natural compounds that enhance plant growth and plant nutrient use efficiency. In this study, biostimulant effects of humic substances (HS) extracted from leonardites were analysed on the metabolism of maize plants grown in hydroponic conditions.

Methods

HS extracted from four leonardites were tested for their auxin-like and gibberellin-like activities. Then, 11 day old maize seedlings were treated for 48 h with five concentrations (0, 0.1, 0.5, 1, and 10 mg C L^?1) of HS. After sampling, root growth and morphology, glutamine synthetase (GS) activity, glutamate synthase (GOGAT) activity, total protein content, soluble sugars content, phenylalanine ammonia-lyase (PAL) activity, soluble phenols, and free phenolic acids were analysed.

Results

HS from leonardites had similar spectroscopic pattern, with small differences. The HS from the South Dakota lignite (HS_USA) had more carboxylic groups, whereas the three from Turkish mines had more aromatic and aliphatic structures. HS_USA best enhanced total root growth, root surface area, and proliferation of secondary roots. Plant nutrient use efficiency was enhanced by HS_4, HS_USA and HS_B, with increment of GS and GOGAT enzymes activity and total protein production. HS stimulated also PAL enzyme activity, followed by a higher production of total soluble phenols, p-hydroxybenzoic acid, p-coumarilic acid, and chlorogenic acid.

Conclusion

This study found that, although the activity of the HS depended on the origin of the leonardite, these compounds can be attributed to the biostimulant products, eliciting plant growth, nitrogen metabolism, and accumulation of phenolic substances.