Quantitative and dynamic expression profile of premature and active forms of the regional ADAM proteins during chicken brain development

The ADAM (A Disintegrin and Metalloprotease) family of transmembrane proteins plays important roles in embryogenesis and tissue formation based on their multiple functional domains. In the present study, for the first time, the expression patterns of the premature and the active forms of six members of the ADAM proteins — ADAM9, ADAM10, ADAM12, ADAM17, ADAM22 and ADAM23 — in distinct parts of the developing chicken brain were investigated by quantitative Western blot analysis from embryonic incubation day (E) 10 to E20. The results show that the premature and the active forms of various ADAM proteins are spatiotemporally regulated in different parts of the brain during development, suggesting that the ADAMs play a very important role during embryonic development.     

Long-term survival and concomitant gene expression of ribozyme-transduced CD4+ T-lymphocytes in HIV-infected patients

BACKGROUND: An anti-HIV-1 tat ribozyme, termed Rz2, has been shown to inhibit HIV-1 infection/replication and to decrease HIV-1-induced pathogenicity in T-lymphocyte cell lines and normal peripheral blood T-lymphocytes. We report here the results of a phase I gene transfer clinical trial using Rz2. METHODS: Apheresis was used to obtain a peripheral blood cell population from each of four HIV-negative donors. After enrichment for CD4+ T-lymphocytes, ex vivo expansion and genetic manipulation (approximately equal aliquots of the cells were transduced with the ribozyme-containing (RRz2) and the control (LNL6) retroviral vector), these cells were infused into the corresponding HIV-1-positive twin recipient. Marking was assessed over an initial 24-week period and in total over an approximate 4-year period. RESULTS: The gene transfer procedure was shown to be safe, and technically feasible. Both RRz2- and LNL6-gene-containing peripheral blood mononuclear cells (PBMC) were detected at all time points examined to 4 years. There was concomitant gene construct expression in the absence of the need for ex vivo peripheral blood cell stimulation and there was no evidence of immune elimination of the neoR T-lymphocytes nor of silencing of the Moloney murine leukemia virus long terminal repeat. CONCLUSIONS: The proof of principle results reported here demonstrate safety and feasibility of this type of gene transfer approach. While not specifically tested, T-lymphocytes containing an anti-HIV gene construct may impact on HIV-1 viral load and CD4+ T-lymphocyte count, potentially representing a new therapeutic modality for HIV-1 infection.     

pIQPNNI: parallel reconstruction of large maximum likelihood phylogenies

SUMMARY: IQPNNI is a program to infer maximum-likelihood phylogenetic trees from DNA or protein data with a large number of sequences. We present an improved and MPI-parallel implementation showing very good scaling and speed-up behavior.     

Genome-wide screening for gene function using RNAi in mammalian cells

Mammalian genome sequencing has identified numerous genes requiring functional annotation. The discovery that dsRNA can direct gene-specific silencing in both model organisms and mammalian cells through RNA interference (RNAi) has provided a platform for dissecting the function of independent genes. The generation of large-scale RNAi libraries targeting all predicted genes within mouse, rat and human cells, combined with the large number of cell-based assays, provides a unique opportunity to perform high-throughput genetics in these complex cell systems. Many different formats exist for the generation of genome-wide RNAi libraries for use in mammalian cells. Furthermore, the use of these libraries in either genetic screens or genetic selections allows for the identification of known and novel genes involved in complex cellular phenotypes and biological processes, some of which underpin human disease. In this review, we examine genome-wide RNAi libraries used in model organisms and mammalian cells and provide examples of how these information rich reagents can be used for determining gene function, discovering novel therapeutic targets and dissecting signalling pathways, cellular processes and complex phenotypes.     

Detritus as a potential food source for protozoans: utilization of fine particulate plant detritus by a heterotrophic flagellate, <Emphasis Type="Italic">Chilomonas paramecium</Emphasis>, and a ciliate, <Emphasis Type="Italic">Tetrahymena pyriformis</Emphasis>

We investigated the direct utilization of fine particulate detritus (dried and homogenized plant material in the size range of bacteria) as a food source for protozoans using axenic cultures of the cryptomonad, heterotrophic flagellate, Chilomonas paramecium, and the hymenostome ciliate, Tetrahymena pyriformis. When fed media containing only particulate detritus, these species revealed growth rates similar to those reported for field populations. The growth rates of Chilomonas fed exclusively particulate detritus were similar to those obtained on a bacterial diet. Considering the high percentage of detritus particles in the size range of bacteria in lakes, our results imply that direct utilization of detritus by protozoans may form an additional pathway of carbon in aquatic food webs that has generally been overlooked.     

Dynamic cellular automata: an alternative approach to cellular simulation

A wide variety of approaches, ranging from Petri nets to systems of partial differential equations, have been used to model very specific aspects of cellular or biochemical functions. Here we describe how an agent-based or dynamic cellular automata (DCA) approach can be used as a very simple, yet very general method to model many different kinds of cellular or biochemical processes. Specifically, using simple pairwise interaction rules coupled with random object moves to simulate Brownian motion, we show how the DCA approach can be used to easily and accurately model diffusion, viscous drag, enzyme rate processes, metabolism (the Kreb's cycle), and complex genetic circuits (the repressilator). We also demonstrate how DCA approaches are able to accurately capture the stochasticity of many biological processes. The success and simplicity of this technique suggests that many other physical properties and significantly more complicated aspects of cellular behavior could be modeled using DCA methods. An easy-to-use, graphically-based computer program, called SimCell, was developed to perform the DCA simulations described here. It is available at http://wishart.biology.ualberta.ca/SimCell/.     

Production of Perennial Vegetation in an Oasis-desert Transition Zone in NW China - Allometric Estimation,and Assessment of Flooding and Use Effects

River oases at the southern fringe of the Taklamakan desert in NW China are surrounded by belts of spontaneous vegetation that protect the oases from sand drift. As an important source of forage, fuel and construction wood, this foreland vegetation is also a component part of the agricultural system of the oases but has been, and still is, destroyed through overuse. Within a broader study that aimed to provide a basis for a sustainable management of this foreland vegetation, biomass and production were studied in four vegetation types dominated either by Alhagi sparsifolia, Calligonum caput-medusae, Populus euphratica, or Tamarix ramosissima that were thought to occur under different regimes of natural flooding in the foreland of Qira (Cele) oasis, Xinjiang, NW China. Shoot biomass components were closely correlated to basal area (Calligonum, Populus, Tamarix) or shrub volume and projection area (Alhagi), enabling non-destructive estimation of stand biomass from shoot diameters or shrub dimensions with sufficient precision using allometric regression equations. Relationships between shoot basal area and biomass of the woody species (Calligonum, Populus and Tamarix) agreed with predictions by a theoretical model of plant vascular systems, suggesting that they are determined by hydraulic and mechanical requirements for shoot architecture. Average aboveground biomass densities of typical stands in late summer were 2.97 Mg/ha in Alhagi, 3.6 Mg/ha in a row plantation and 10.9 Mg/ha in homogenous stands of Calligonum, 22–29 Mg/ha in 22 year-old Populus forests and 1.9–3.1 Mg/ha in Tamarix-dominated vegetation. Annual aboveground production including wood and assimilation organs ranged from 2.11 to 11.3 Mg/ha in plantations of Calligonum, 3.17 to 6.12 Mg/ha in Populus, and 1.55 to 1.74 Mg/ha (based on total ground area) or 3.10 to 7.15 Mg/ha (in homogenous stands) in Tamarix. Production of Alhagi is equal to peak biomass. A thinning treatment simulating use by the local population enhanced productivity of Calligonum, Populus and Tamarix. A complete harvest of Alhagi in late August decreased production in the following year. An artificial flood irrigation treatment did not sufficiently increase soil water content except in the uppermost layer and had no clear beneficial effect on growth of the four species and even a negative effect on Alhagi, which was due to increased competition from annual species. As biomass and production with or without artificial irrigation were much higher than values expected for rain-fed desert vegetation at a mean annual precipitation of 35 mm, it is concluded that the existence of all vegetation types studied is probably based on permanent access to groundwater and that natural floods or precipitation do not contribute to their water supply. The effects of agricultural groundwater use in the oasis on groundwater in the foreland of the oasis need further study. Sustainable use of this productive vegetation is possible but requires proper management.     

PRAM-1 potentiates arsenic trioxide-induced JNK activation

The promyelocytic leukemia RARalpha target gene encoding an adaptor molecule-1 (PRAM-1) is involved in a signaling pathway induced by retinoic acid in acute promyelocytic leukemia (APL) cells. To better understand the function of PRAM-1, we have undertaken the identification of its partners through a yeast two-hybrid screen. Here, we show that the proline-rich domain of PRAM-1 interacted with the Src homology 3 (SH3) domain of hematopoietic progenitor kinase 1 (HPK-1)-interacting protein of 55 kDa (HIP-55, also called SH3P7 and Abp1) known to stimulate the activity of HPK-1 and c-Jun N-terminal kinase (JNK). Overexpression of PRAM-1 in the NB4 APL cell line increased arsenic trioxide-induced JNK activation through a caspase 3-like-dependent activity. Dissociation of the SH3 domain from the rest of the HIP-55 protein was observed in the NB4 APL cell line treated with arsenic trioxide due to specific cleavage by caspase 3-like enzymes. The cleavage of HIP-55 correlated with the induction of PRAM-1 mRNA and protein expression. Taken together, our results suggest that the caspase 3-cleaved SH3 domain of HIP-55 is likely involved in PRAM-1-mediated JNK activation upon arsenic trioxide-induced differentiation of NB4 cells.     

Genomic heterogeneity of background substitutional patterns in Drosophila melanogaster

Mutation is the underlying force that provides the variation upon which evolutionary forces can act. It is important to understand how mutation rates vary within genomes and how the probabilities of fixation of new mutations vary as well. If substitutional processes across the genome are heterogeneous, then examining patterns of coding sequence evolution without taking these underlying variations into account may be misleading. Here we present the first rigorous test of substitution rate heterogeneity in the Drosophila melanogaster genome using almost 1500 nonfunctional fragments of the transposable element DNAREP1_DM. Not only do our analyses suggest that substitutional patterns in heterochromatic and euchromatic sequences are different, but also they provide support in favor of a recombination-associated substitutional bias toward G and C in this species. The magnitude of this bias is entirely sufficient to explain recombination-associated patterns of codon usage on the autosomes of the D. melanogaster genome. We also document a bias toward lower GC content in the pattern of small insertions and deletions (indels). In addition, the GC content of noncoding DNA in Drosophila is higher than would be predicted on the basis of the pattern of nucleotide substitutions and small indels. However, we argue that the fast turnover of noncoding sequences in Drosophila makes it difficult to assess the importance of the GC biases in nucleotide substitutions and small indels in shaping the base composition of noncoding sequences.     

Tubulin assembly, taxoid site binding, and cellular effects of the microtubule-stabilizing agent dictyostatin

(-)-Dictyostatin is a sponge-derived, 22-member macrolactone natural product shown to cause cells to accumulate in the G2/M phase of the cell cycle, with changes in intracellular microtubules analogous to those observed with paclitaxel treatment. Dictyostatin also induces assembly of purified tubulin more rapidly than does paclitaxel, and nearly as vigorously as does dictyostatin's close structural congener, (+)-discodermolide (Isbrucker et al. (2003), Biochem. Pharmacol. 65, 75-82). We used synthetic (-)-dictyostatin to study its biochemical and cytological activities in greater detail. The antiproliferative activity of dictyostatin did not differ greatly from that of paclitaxel or discodermolide. Like discodermolide, dictyostatin retained antiproliferative activity against human ovarian carcinoma cells resistant to paclitaxel due to beta-tubulin mutations and caused conversion of cellular soluble tubulin pools to microtubules. Detailed comparison of the abilities of dictyostatin and discodermolide to induce tubulin assembly demonstrated that the compounds had similar potencies. Dictyostatin inhibited the binding of radiolabeled discodermolide to microtubules more potently than any other compound examined, and dictyostatin and discodermolide had equivalent activity as inhibitors of the binding of both radiolabeled epothilone B and paclitaxel to microtubules. These results are consistent with the idea that the macrocyclic structure of dictyostatin represents the template for the bioactive conformation of discodermolide.