Role of the duplicated CCAAT box region in gamma-globin gene regulation and hereditary persistence of fetal haemoglobin.

Hereditary persistence of fetal haemoglobin (HPFH) is a clinically important condition in which a change in the developmental specificity of the gamma-globin genes results in varying levels of expression of fetal haemoglobin in the adult. The condition is benign and can significantly alleviate the symptoms of thalassaemia or sickle cell anaemia when co-inherited with these disorders. We have examined structure-function relationships in the -117 HPFH gamma promoter by analysing the effect of mutating specific promoter elements on the functioning of the wild-type and HPFH promoters. We find that CCAAT box mutants dramatically affect expression from the HPFH promoter in adult blood but have little effect on embryonic/fetal expression from the wild-type promoter. Our results suggest that there are substantial differences in the structure of the wild-type gamma promoter expressed early in development and the adult HPFH promoter. Together with previous results, this suggests that gamma silencing is a complex multifactorial phenomenon rather than being the result of a simple repressor binding to the promoter. We present a model for gamma-globin gene silencing that has significant implications for attempts to reactivate the gamma promoters in human adults by pharmacological means.     

Identification of novel genes and networks governing hematopoietic stem cell development

Hematopoietic stem cells (HSCs) are capable of giving rise to all blood cell lineages throughout adulthood, and the generation of engraftable HSCs from human pluripotent stem cells is a major goal for regenerative medicine. Here, we describe a functional genome‐wide RNAi screen to identify genes required for the differentiation of embryonic stem cell (ESC) into hematopoietic stem/progenitor cells (HSPCs) in vitro. We report the discovery of novel genes important for the endothelial‐to‐hematopoietic transition and subsequently for HSPC specification. High‐throughput sequencing and bioinformatic analyses identified twelve groups of genes, including a set of 351 novel genes required for HSPC specification. As in vivo proof of concept, four of these genes, Ap2a1, Mettl22, Lrsam1, and Hal, are selected for validation, confirmed to be essential for HSPC development in zebrafish and for maintenance of human HSCs. Taken together, our results not only identify a number of novel regulatory genes and pathways essential for HSPC development but also serve as valuable resource for directed differentiation of therapy grade HSPCs using human pluripotent stem cells.     

TLR Agonist Augments Prophylactic Potential of Acid Inducible Antigen Rv3203 against Mycobacterium tuberculosis H37Rv in Experimental Animals

In general, the members of Lip gene family of Mycobacterium tuberculosis evoke strong immune response in the host. Keeping this fact into consideration, we investigated role of Rv3203, a cell wall associated protein with lipolytic activity, in imparting protection against experimental murine tuberculosis. The data of the present study suggested that archaeosome encapsulated Rv3203 induce strong lymphocyte proliferation, up-regulated Th-1 biased cytokines profile, increased expression of co-stimulatory markers on both antigen presenting cells and T lymphocytes. The immuno-prophylactic response was further modulated by exposure of the animals to zymosan, a TLR2/6 agonist, prior to immunization with archaeosome encapsulated Rv3203. Interestingly, pre-treatment of experimental animals with zymosan boosted strong immunological memory as compared to archaeosome encapsulated Rv3203 as well as BCG vaccine. We conclude that priming of immunized animal with TLR agonist followed by immunization with archaeosomes encapsulated Rv3203 offer substantial protection against tuberculosis infection and could be a potential subunit vaccine based prophylactic strategy.     

Isolation and characterisation of cDNA clones for the A alpha- and gamma-chains of human fibrinogen.

cDNA clones coding for the A alpha- and gamma-chains of human fibrinogen have been isolated from an adult liver cDNA library. Clones were identified by hybridisation with mixtures of synthetic oligonucleotides 17 bases long, predicted using amino acid sequence data for each chain. The cDNA insert sizes are 1,950bp for A alpha-fibrinogen and 950bp for gamma-fibrinogen. The clones do not show any cross-hybridisation. Each cDNA hybridises to a unique sequence in the human genome. In adult human liver, Northern blots give an estimated messenger RNA size of 2.6kb for A alpha-fibrinogen and 1.8kb for gamma-fibrinogen.     

Biological breakdown of benzylpenicillin by preformed mats of penicillin-producing organisms

1. Penicillium chrysogenum and Aspergillus flavus degraded benzylpenicillin in the same way. 2. Degradation of the antibiotic was initiated by the cleavage of phenylacetic acid from 6-aminopenicillanic acid. 3. Phenylacetic acid was left unchanged whereas 6-aminopenicillanic acid was degraded into cysteine and valine. This reaction is probably complex. 4. Cysteine was not utilized but valine was cleaved into acetone and glycine. Catabolism of valine by the preformed mats of the two moulds confirms this result. This step probably involves the formation of propan-2-ol. 5. Cleavage of benzylpenicillin into phenylacetic acid and 6-aminopenicillanic acid was performed through the activity of a cellular-bound enzyme.     

CYTOGENETIC STUDIES IN CLARKIA,SECTION PRIMIGENIA. II. A CYTOLOGICAL SURVEY OF CLARKIA ARCUATA AND CLARKIA LASSENENSIS

Clarkia arcuata and C. lassenensis are the 2 members of the subsection Flexicaules. Although closely related morphologically, they show very different patterns of chromosomal variability in nature. About 25% of the plants grown from wild seed of C. arcuata, a predominantly cross-pollinating species, were heterozygous for 1 or 2 translocations; such heterozygotes were found in 5 of the 9 populations sampled. An analysis of the chromosome pairing in intraspecific crosses indicated that at least 5 different translocations giving a ring of 4 with the “standard” strain, 2 giving a ring of 6, and 2 giving a ring of 8 are present in nature. No arrangement was found with widespread distribution, and it is impossible to say at present what might be the primitive arrangement of this species. One population was found to contain an inversion, a rearrangement which is very rare in Clarkia at the intraspecific level. In C. lassenensis, a predominantly self-pollinating species, only 6% (3 plants) of a sample of 53 were translocation heterozygotes, and these heterozygotes were found in only 2 of 13 populations. Intraspecific crosses indicated that one chromosome arrangement, the “standard,” was present throughout the species range.     

Identification and characterization of Neopestalotiopsis clavispora associated with leaf blight of small cardamom (Elettaria cardamomum Maton)

Leaf blight is a major foliar disease prevalent in all cardamom‐cultivating tracts, manifesting in diverse forms of symptoms. In this study, six symptomatological variants were delineated based on the expression of foliar symptoms in cardamom genotypes (Malabar, Mysore and Vazhukka) and designated as SV 1 to SV 6. Among the symptomatological variants, SV 1, SV 2, SV 3 and SV 6 were more pronounced in Vazhukka, while SV 4 and SV 5 were prominent in Malabar type. Subsequent isolation from the variants yielded whitish colonies, which were correspondingly coded as SV 1 to SV 6. The conidia were fusiform, five‐celled, with three median versicoloured cells, two terminal hyaline cells and measured 23.1–27.25 × 3.84–4.43 μm. The apical cells had two to three tubular, flexuous, unbranched appendages, whereas the basal appendage was single, tubular and unbranched. Based on conidial characteristics and molecular characterization with internal transcribed spacer rDNA region, partial β‐tubulin, translation elongation factor 1 alpha and large subunit (28S) of the nrRNA genes revealed identity of the pathogens as Neopestalotiopsis clavispora. The pathogenicity test was performed on Malabar, Mysore and Vazhukka genotypes, and Koch’s postulates were proved. In‐vitro interaction at three temperature regimes indicated that N. clavispora was inhibitory to Colletotrichum gloeosporioides at 10 and 30°C. Among the fungicides, carbendazim, propiconazole and carbendazim‐mancozeb completely arrested hyphal growth of N. clavispora under in‐vitro conditions. This study constitutes first report on the association of Neopestalotiopsis clavispora with leaf blight disease of small cardamom.     

Stochastic simulations on the reliability of action potential propagation in thin axons

It is generally assumed that axons use action potentials (APs) to transmit information fast and reliably to synapses. Yet, the reliability of transmission along fibers below 0.5 μm diameter, such as cortical and cerebellar axons, is unknown. Using detailed models of rodent cortical and squid axons and stochastic simulations, we show how conduction along such thin axons is affected by the probabilistic nature of voltage-gated ion channels (channel noise). We identify four distinct effects that corrupt propagating spike trains in thin axons: spikes were added, deleted, jittered, or split into groups depending upon the temporal pattern of spikes. Additional APs may appear spontaneously; however, APs in general seldom fail (<1%). Spike timing is jittered on the order of milliseconds over distances of millimeters, as conduction velocity fluctuates in two ways. First, variability in the number of Na channels opening in the early rising phase of the AP cause propagation speed to fluctuate gradually. Second, a novel mode of AP propagation (stochastic microsaltatory conduction), where the AP leaps ahead toward spontaneously formed clusters of open Na channels, produces random discrete jumps in spike time reliability. The combined effect of these two mechanisms depends on the pattern of spikes. Our results show that axonal variability is a general problem and should be taken into account when considering both neural coding and the reliability of synaptic transmission in densely connected cortical networks, where small synapses are typically innervated by thin axons. In contrast we find that thicker axons above 0.5 μm diameter are reliable.     

Induction of uPA gene expression by the blockage of E-cadherin via Src- and Shc-dependent Erk signaling

Loss of E-cadherin-mediated cell-cell adhesion and expression of proteolytic enzymes characterize the transition from benign lesions to invasive, metastatic tumor, a rate-limiting step in the progression from adenoma to carcinoma in vivo. A soluble E-cadherin fragment found recently in the serum and urine of cancer patients has been shown to disrupt cell-cell adhesion and to drive cell invasion in a dominant-interfering manner. Physical disruption of cell-cell adhesion can be mimicked by the function-blocking antibody Decma. We have shown previously in MCF7 and T47D cells that urokinase-type plasminogen activator (uPA) activity is up-regulated upon disruption of E-cadherin-dependent cell-cell adhesion. We explored the underlying molecular mechanisms and found that blockage of E-cadherin by Decma elicits a signaling pathway downstream of E-cadherin that leads to Src-dependent Shc and extracellular regulated kinase (Erk) activation and results in uPAgene activation. siRNA-mediated knockdown of endogenous Src-homology collagen protein (Shc) and subsequent expression of single Shc isoforms revealed that p46(Shc) and p52(Shc) but not p66(Shc) were able to mediate Erk activation. A parallel pathway involving PI3K contributed partially to Decma-induced Erk activation. This report describes that disruption of E-cadherin-dependent cell-cell adhesion induces intracellular signaling with the potential to enhance tumorigenesis and, thus, offers new insights into the pathophysiological mechanisms of tumor development.