Towards β-globin gene-targeting with integrase-defective lentiviral vectors

We have developed an integrase-defective lentiviral (LV) vector in combination with a gene-targeting approach for gene therapy of β-thalassemia. The β-globin gene-targeting construct has two homologous stems including sequence upstream and downstream of the β-globin gene, a β-globin gene positioned between hygromycin and neomycin resistant genes and a herpes simplex virus type 1 thymidine kinase (HSVtk) suicide gene. Utilization of integrase-defective LV as a vector for the β-globin gene increased the number of selected clones relative to non-viral methods. This method represents an important step toward the ultimate goal of a clinical gene therapy for β-thalassemia.     

Features analysis for identification of date and party hubs in protein interaction network of <Emphasis Type="Italic">Saccharomyces Cerevisiae</Emphasis>

Background  

It has been understood that biological networks have modular organizations which are the sources of their observed complexity. Analysis of networks and motifs has shown that two types of hubs, party hubs and date hubs, are responsible for this complexity. Party hubs are local coordinators because of their high co-expressions with their partners, whereas date hubs display low co-expressions and are assumed as global connectors. However there is no mutual agreement on these concepts in related literature with different studies reporting their results on different data sets. We investigated whether there is a relation between the biological features of Saccharomyces Cerevisiae 's proteins and their roles as non-hubs, intermediately connected, party hubs, and date hubs. We propose a classifier that separates these four classes.     

Weak organic acid stress inhibits aromatic amino acid uptake by yeast, causing a strong influence of amino acid auxotrophies on the phenotypes of membrane transporter mutants.

The ability of yeasts to grow in the presence of weak organic acid preservatives is an important cause of food spoilage. Many of the determinants of acetate resistance in Saccharomyces cerevisiae differ from the determinants of resistance to the more lipophilic sorbate and benzoate. Interestingly, we show in this study that hypersensitivity to both acetate and sorbate results when the cells have auxotrophic requirements for aromatic amino acids. In tryptophan biosynthetic pathway mutants, this weak acid hypersensitivity is suppressed by supplementing the medium with high levels of tryptophan or, in the case of sorbate sensitivity, by overexpressing the Tat2p high affinity tryptophan permease. Weak acid stress therefore inhibits uptake of aromatic amino acids from the medium. This allows auxotrophic requirements for these amino acids to strongly influence the resistance phenotypes of mutant strains. This property must be taken into consideration when using these phenotypes to attribute functional assignments to genes. We show that the acetate sensitivity phenotype previously ascribed to yeast mutants lacking the Pdr12p and Azr1p plasma membrane transporters is an artefact arising from the use of trp1 mutant strains. These transporters do not confer resistance to high acetate levels and, in prototrophs, their presence is actually detrimental for this resistance.     

Characterization of ISXax1, a novel insertion sequence restricted to Xanthomonas axonopodis pv. phaseoli (variants fuscans and non-fuscans) and Xanthomonas axonopodis pv. vesicatoria

ISXax1 is a novel insertion sequence belonging to the IS256 and Mutator families. Dot blot, Southern blot, and PCR analyses revealed that ISXax1 is restricted to Xanthomonas axonopodis pv. phaseoli (variants fuscans and non-fuscans) and X. axonopodis pv. vesicatoria strains. Directed AFLP also showed that a high degree of polymorphism is associated with ISXax1 insertion in these strains.     

Molecular characterization of Cryptosporidium isolates from humans and animals in Iran

Isolates of Cryptosporidium spp. from human and animal hosts in Iran were characterized on the basis of both the 18S rRNA gene and the Laxer locus. Three Cryptosporidium species, C. hominis, C. parvum, and C. meleagridis, were recognized, and zoonotically transmitted C. parvum was the predominant species found in humans.     

Hog1 mitogen-activated protein kinase phosphorylation targets the yeast Fps1 aquaglyceroporin for endocytosis, thereby rendering cells resistant to acetic acid

Aquaporins and aquaglyceroporins form the membrane channels that mediate fluxes of water and small solute molecules into and out of cells. Eukaryotes often use mitogen-activated protein kinase (MAPK) cascades for the intracellular signaling of stress. This study reveals an aquaglyceroporin being destabilized by direct MAPK phosphorylation and also a stress resistance being acquired through this channel loss. Hog1 MAPK is transiently activated in yeast exposed to high, toxic levels of acetic acid. This Hog1 then phosphorylates the plasma membrane aquaglyceroporin, Fps1, a phosphorylation that results in Fps1 becoming ubiquitinated and endocytosed and then degraded in the vacuole. As Fps1 is the membrane channel that facilitates passive diffusional flux of undissociated acetic acid into the cell, this loss downregulates such influx in low-pH cultures, where acetic acid (pKa, 4.75) is substantially undissociated. Consistent with this downregulation of the acid entry generating resistance, sensitivity to acetic acid is seen with diverse mutational defects that abolish endocytic removal of Fps1 from the plasma membrane (loss of Hog1, loss of the soluble domains of Fps1, a T231A S537A double mutation of Fps1 that prevents its in vivo phosphorylation, or mutations generating a general loss of endocytosis of cell surface proteins [doa4Delta and end3Delta]). Remarkably, targetting of Fps1 for degradation may be the major requirement for an active Hog1 in acetic acid resistance, since Hog1 is largely dispensable for such resistance when the cells lack Fps1. Evidence is presented that in unstressed cells, Hog1 exists in physical association with the N-terminal cytosolic domain of Fps1.     

Investigation on Mitochondrial tRNA<Superscript>Leu/Lys</Superscript>, NDI and ATPase 6/8 in Iranian Multiple Sclerosis Patients

As with chromosomal DNA, the mitochondrial DNA (mtDNA) can contain mutations that are highly pathogenic .In fact, many diseases of the central nervous system are known to be caused by mutations in mtDNA. Dysfunction of the mitochondrial Respiratory Chain (RC) has been shown in patients with neurological disease including Alzheimer’s disease (AD), Parkinson’s disease (PD) and Multiple sclerosis (MS). MS is a demyelinating disease of central nervous system characterized by morphological hallmarks of inflammation, demyelination and axonal loss. Considering this importance, we decided to investigate several highly mutative parts of mtDNA for point mutations as MT-LTI (tRNA^{Leucine1(UUA/G)}), MT-NDI (NADH Dehydrogenase subunit 1), MT-COII (Cytochrome c oxidase subunit II), MT-TK (tRNA^Lysine), MT-ATP8 (ATP synthase subunit F0 8) and MT-ATP6 (ATP synthase subunit F0 6) in 20 Iranian MS patients and 80 age-matched control subjects by PCR and automated DNA sequencing to evaluate any probable point mutations. Our results revealed that 15 (75%) out of 20 MS patients had point mutations. Some of point mutations were newly found in this study. This study suggested that point mutation occurred in mtDNA might be involved in pathogenesis of MS.     

Expression of Hoxa2 in cells entering chondrogenesis impairs overall cartilage development

Vertebrate Hox genes act as developmental architects by patterning embryonic structures like axial skeletal elements, limbs, brainstem territories, or neural crest derivatives. While active during the patterning steps of development, these genes turn out to be down-regulated in specific differentiation programs like that leading to chondrogenesis. To investigate why chondrocyte differentiation is correlated to the silencing of a Hox gene, we generated transgenic mice allowing Cre-mediated conditional misexpression of Hoxa2 and induced this gene in Collagen 2 alpha 1-expressing cells committed to enter chondrogenesis. Persistent Hoxa2 expression in chondrogenic cells resulted in overall chondrodysplasia with delayed cartilage hypertrophy, mineralization, and ossification but without proliferation defects. The absence of skeletal patterning anomaly and the regular migration of precursor cells indicated that the condensation step of chondrogenesis was normal. In contrast, closer examination at the differentiation step showed severely impaired chondrocyte differentiation. In addition, this inhibition affected structures independently of their embryonic origin. In conclusion, for the first time here, by a cell-type specific misexpression, we precisely uncoupled the patterning function of Hoxa2 from its involvement in regulating differentiation programs per se and demonstrate that Hoxa2 displays an anti-chondrogenic activity that is distinct from its patterning function.     

Stoichiometric constraints on resource use, competitive interactions, and elemental cycling in microbial decomposers

Heterotrophic microbial decomposers, such as bacteria and fungi, immobilize or mineralize inorganic elements, depending on their elemental composition and that of their organic resource. This fact has major implications for their interactions with other consumers of inorganic elements. We combine the stoichiometric and resource-ratio approaches in a model describing the use by decomposers of an organic and an inorganic resource containing the same essential element, to study its consequences on decomposer interactions and their role in elemental cycling. Our model considers the elemental composition of organic matter and the principle of its homeostasis explicitly. New predictions emerge, in particular, (1) stoichiometric constraints generate a trade-off between the R* values of decomposers for the two resources; (2) they create favorable conditions for the coexistence of decomposers limited by different resources and with different elemental demands; (3) however, combined with conditions on species-specific equilibrium limitation, they draw decomposers toward colimitation by the organic and inorganic resources on an evolutionary time scale. Moreover, we derive the conditions under which decomposers switch from consumption to excretion of the inorganic resource. We expect our predictions to be useful in explaining the community structure of decomposers and their interactions with other consumers of inorganic resources, particularly primary producers.