The Dictyostelium LIM domain-containing protein LIM2 is essential for proper chemotaxis and morphogenesis

We have identified limB, a gene encoding a novel LIM domain-containing protein, LIM2, in a screen for genes required for morphogenesis. limB null cells aggregate, although poorly, but they are unable to undergo morphogenesis, and the aggregates arrest at the mound stage. limB null cells exhibit an aberrant actin cytoskeleton and have numerous F-actin-enriched microspikes. The cells exhibit poor adhesion to a substratum and do not form tight cell-cell agglomerates in suspension. Furthermore, limB null cells are unable to properly polarize in chemoattractant gradients and move very poorly. Expression of limB from a prestalk-specific but not a prespore-specific promoter complements the morphogenetic defects of the limB null strain, suggesting that the limB null cell developmental defect results from an inability to properly sort prestalk cells. LIM2 protein is enriched in the cortex of wild-type cells, although it does not colocalize with the actin cytoskeleton. Our analysis indicates that LIM2 is a new regulatory protein that functions to control rearrangements of the actin cytoskeleton and is required for cell motility and chemotaxis. Our findings may be generally applicable to understanding pathways that control cell movement and morphogenesis in all multicellular organisms. Structure function studies on the LIM domains are presented.     

Effects of Ellagic Acid by Oral Administration on N-Acetylation and Metabolism of 2-Aminofluorene in Rat Brain Tissues

Numerous studies have demonstrated that the Acetyl Coenzyme A-dependent arylamine NAT enzyme exist in many tissues of experimental animals including humans, and that NAT has been shown to be exist in mouse brain tissue. Increased NAT activity levels are associated with increased sensitivity to the mutagenic effects of arylamine carcinogens. Attenuation of liver NAT activity is related to breast and bladder cancer processes. Therefore, the effects of ellagic acid (EA) on the in vitro and in vivo N-acetylation of 2-aminofluorene (AF) were investigated in cerebrum, cerebellum and pineal gland tissues from male Sprague-Dawley rats. For in vitro examination, cytosols with or without EA (0.5–500 M) co-treatment decreased 7–72%, 15–63% and 10–78% of AF acetylation for cerebrum, cerebellum and pineal gland tissues, respectively. For in vivo examination, EA and AF at the same time treated groups with all 3 examined tissues did show significant differences (the changes of total amounts of AF and AF metabolites based on the Anova analysis) when compared to the ones without EA cotreatment rats. The pretreatment of male rats with EA (10 mg/kg) 24 hr prior to the administration of AF (50 mg/kg) (one day of EA administration suffice to induce large changes in phase II enzyme activity) resulted in a 76% decrease in total AF and metabolites in pineal gland but did not show significant differences in cerebrum and cerebellum tissues. This is the first demonstration to show that EA decreases the N-acetylation of carcinogens in rat brain tissues.     

Paeonol promoted 2-aminofluorene and p-aminobenzoic acid acetylations by mononuclear leucocytes from Sprague-Dawley rats

Following exposure of rats to the arylamine carcinogen 2-aminofluorene, DNA-carcinogen adducts were found in the target tissues of the liver and bladder, and also in circulating leucocytes. This work investigated how paeonol affects arylamine (2-aminofluorene and p-aminobenzoic acid) acetylations in rat leucocytes. Evidence is presented showing that rat mononuclear leucocytes are capable of acetylating 2-aminofluorene and p-aminobenzoic acid. Paeonol promoted 2-aminofluorene and p-aminobenzoic acid acetylation. Cultured lymphocytes produced about twice as much N-acetyl-2-aminofluorene from 2-aminofluorene and 2.2-fold as much N-acetyl-p-aminobenzoic acid from p-aminobenzoic acid as monocytes. After cotreatment with paeonol, the lymphocyte and monocyte cultures indicated that paeonol did increase 2-aminofluorene and p-aminobenzoic acid acetylations.     

Effects of luteolin on arylamine N-acetyltransferase activity in human liver tumour cells

The human liver tumour cell line (J5) was selected in order to evaluate whether or not luteolin affected arylamine N-acetyltransferase (NAT) activity. Using high performance liquid chromatography, the NAT activity for acetylation of arylamine substrates (2-aminofluorene and p-aminobenzoic acid) was determined. The cytosolic NAT activity in human liver tumour cells was 2.74+/-0.26 and 1.68+/-0.20 nmol/min/mg of protein for 2-aminofluorene and p-aminobenzoic acid, respectively. Luteolin displayed a dose-dependent inhibition to cytosolic NAT activity and intact human liver tumour cells. Time-course experiments showed that NAT activity measured from intact human liver tumour cells was inhibited by luteolin for up to 24 h. Using standard steady-state kinetic analysis, it was shown that luteolin was a possible noncompetitive inhibitor to NAT activity in cytosols. This report is the first to show how luteolin affects NAT activity in human liver tumour cells.     

The downregulation of Bcl-2 expression is necessary for theophylline-induced apoptosis of eosinophil

Apoptosis of eosinophils is of increasingly important value in modulating allergic inflammatory airway diseases, such as asthma, and is suppressed by interleukin-5 (IL-5) in in vitro culture. In this study, we examined the effects of theophylline on survival/apoptosis, intracellular cAMP concentration, and Bcl-2 protein expression. Treatment with theophylline protected eosinophils against IL-5-mediated inhibition of apoptosis with a simultaneous suppression of survival in a dose-dependent manner. Theophylline caused an increase in the intracellular cAMP levels of IL-5-stimulated eosinophils. Enhancement of eosinophil apoptosis was consistent with an increase in DNA fragmentation in eosinophils treated with theophylline. On the other hand, the Bcl-2 protein appeared to be expressed constitutively in freshly isolated eosinophils. Bcl-2 expression was augmented by IL-5 stimulation, yet it was considerably inhibited by theophylline treatment. These data suggest that intracellular cAMP levels and Bcl-2 expression are involved in the suppression of eosinophil survival by theophylline.     

Transforming growth factor-beta induces apoptosis in activated murine T cells through the activation of caspase 1-like protease

Transforming growth factor-beta (TGF-beta) has been known as a potent immunosuppressive cytokine that can induce apoptosis in lymphoid cells. We established an IL-2-independent cell line, CTLL-2A, from murine T cell line CTLL-2. CTLL-2A expressed higher levels of CD95, CD69, and CD18 molecules than CTLL-2 did, suggesting a more activated state in CTLL-2A than in the CTLL-2 by phenotype. Exposing both CTLL-2 and CTLL-2A to TGF-beta results in differential apoptosis patterns defined by DNA fragmentation and plasma membrane alteration. Among the bcl-2 family members, bcl-2, bcl-w, and bcl-x(L) were also differently expressed in these two cell lines. In CTLL-2A, bcl-x(L) was amplified as a major anti-apoptotic molecule, and TGF-beta-induced cell death was more enhanced than in the original cell line. Caspase 1-like protease was activated by TGF-beta treatment and consequently it cleaved bcl-x(L) in CTLL-2A. TGF-beta-induced DNA fragmentation and cleavage of bcl-x(L) were inhibited by pretreatment with tetra peptide caspase 1 inhibitor, YVAD.cmk. These findings suggest that TGF-beta induces cell death in activated murine T cells through cleavage of bcl-x(L) via activated caspase 1-like protease, which may act as an important executor in that process.     

Light chain of natural antibody plays a dominant role in protein antigen binding

Examinations of the contribution and the specificity of heavy (H) and light (L) chains of natural antibodies to antigen binding may help us to better understand antigen recognition and the development of naive B cells. We previously generated natural Fab antibody fragments reactive to preS1 of HBV using a naive, non-immunized Fab antibody library derived from peripheral B cells of a normal healthy volunteer. We now constructed expression vectors for the Fd (VH + CH1), L chain, and scFv fragments using the sequences encoding parental Fabs as a source of natural antibody genes. The recombinant antibody fragments were expressed as inclusion bodies in E. coli BL21 (DE) cells. When denatured and then refolded, the antibody fragments retained their binding properties. Recombinant L chains and scFvs exhibited three- to 40-fold higher affinities (in the order of 10(7) M(-1)) over the parental Fabs, whereas the affinities of Fds (in the order of 10(5) M(-1)) were much lower compared to the parental Fabs. The results obtained from sandwich ELISA revealed that the L chains bound the virus more efficiently than Fds. Additional experiments were performed to evaluate the specificity of the recombinant fragments for surface proteins of HBV. Fds and L chains were reactive towards HBsAg and the preS2 peptide as well as preS1 and showed patterns of epitope recognition quite different from those of parental Fabs. The data presented here demonstrate that the prominence of the L chain in determining protein binding activity is a property of natural antibodies and is quite unlike the antibodies induced by immunization, and that the specificity of Fab is not determined by the individual antibody chain but by the correct pairing of H and L chain.     

Tests of continuum theories as models of ion channels. I. Poisson-Boltzmann theory versus Brownian dynamics

Continuum theories of electrolytes are widely used to describe physical processes in various biological systems. Although these are well-established theories in macroscopic situations, it is not clear from the outset that they should work in small systems whose dimensions are comparable to or smaller than the Debye length. Here, we test the validity of the mean-field approximation in Poisson-Boltzmann theory by comparing its predictions with those of Brownian dynamics simulations. For this purpose we use spherical and cylindrical boundaries and a catenary shape similar to that of the acetylcholine receptor channel. The interior region filled with electrolyte is assumed to have a high dielectric constant, and the exterior region representing protein a low one. Comparisons of the force on a test ion obtained with the two methods show that the shielding effect due to counterions is overestimated in Poisson-Boltzmann theory when the ion is within a Debye length of the boundary. As the ion gets closer to the boundary, the discrepancy in force grows rapidly. The implication for membrane channels, whose radii are typically smaller than the Debye length, is that Poisson-Boltzmann theory cannot be used to obtain reliable estimates of the electrostatic potential energy and force on an ion in the channel environment.     

Design of metabolic feed controllers: application to high-density fermentations of Pichia pastoris

High-density cultures of the methylotrophic yeast Pichia pastoris were found to exhibit oscillatory metabolic behavior when fed methanol under closed-loop operations using a dissolved oxygen-based bioreactor feed controller (DOstat). This behavior, if left unattended, led to the irreversible loss of culture productivity, 1 to 2 days after growth on methanol commenced, presumably through the accumulation of incompletely oxidized intermediates. To provide insights into how fermentation operation conditions and strain variations might contribute to this phenomenon a theoretical study was initiated. In this article, a simple mathematical model of the closed-loop DOstat is developed and analyzed with the goal of deriving theoretical stability criteria applicable to the design of metabolic feed controllers during high-cell-density fermentations. The model consists of a system of differential, integral, and algebraic equations describing the biological process and the components of the standard proportional-integral (PI) feedback controller. Inputs into the process model include metabolic pathway information, oxidative metabolism stoichiometry, and substrate uptake kinetics. Frequency-response analysis and the Bode stability criterion are applied to derive controller stability criteria with particular emphasis on elucidating the role(s) that model parameters, both biological and operational, have on system stability. The results of this analysis are used to construct a closed-loop fermentation-operating diagram relating fermentation operating parameters to the oxidative capacity of the culture. The utility of this analysis is demonstrated through the application of the results to the design and stabilization of the DOstat during high-density fermentations of P. pastoris growing on methanol or glycerol. From this analysis, it is possible to conclude that, when the rate of oxygen transfer approaches in magnitude the rate of oxygen utilization, the potential for controller destabilization is greatest. Under these conditions, the region of parameter space associated with stable controller operations is further constrained. Because the only system-specific experimental inputs into the model are the routinely measured residual substrate and dissolved oxygen concentrations, the framework presented should provide simple and practical theoretical guidelines relevant to the design of similar industrial fermentation feed controllers independent of the specifics of the biological system at hand.     

The diversity of beetle assemblages in different habitat types in Sabah, Malaysia

The diversity of beetle assemblages in different habitat types (primary forest, logged forest, acacia plantation and oil palm plantation) in Sabah, Malaysia was investigated using three different methods based on habitat levels (Winkler sampling, flight-interception-trapping and mist-blowing). The overall diversity was extremely high, with 1711 species recorded from only 8028 individuals and 81 families (115 family and subfamily groups). Different degrees of environmental changes had varying effects on the beetle species richness and abundance, with oil palm plantation assemblage being most severely affected, followed by acacia plantation and then logged forest. A few species became numerically dominant in the oil palm plantation. In terms of beetle species composition, the acacia fauna showed much similarity with the logged forest fauna, and the oil palm fauna was very different from the rest. The effects of environmental variables (number of plant species, sapling and tree densities, amount of leaf litter, ground cover, canopy cover, soil pH and compaction) on the beetle assemblage were also investigated. Leaf litter correlated with species richness, abundance and composition of subterranean beetles. Plant species richness, tree and sapling densities correlated with species richness, abundance and composition of understorey beetles while ground cover correlated only with the species richness and abundance of these beetles. Canopy cover correlated only with arboreal beetles. In trophic structure, predators represented more than 40% of the species and individuals. Environmental changes affected the trophic structure with proportionally more herbivores (abundance) but fewer predators (species richness and abundance) in the oil palm plantation. Biodiversity, conservation and practical aspects of pest management were also highlighted in this study.