Granulocyte colony-stimulating factor: molecular mechanisms of action during steady state and 'emergency' hematopoiesis

Neutrophils are phagocytes whose principal function is to maintain anti-bacterial immunity. Neutrophils ingest and kill invading bacteria, releasing cytotoxic, chemotactic and inflammatory mediators at sites of infection. This serves to control the immediate host immune response and attract other cells, such as macrophages and dendritic cells, which are important for establishing long-term adaptive immunity. Neutrophils thus contribute to both the initiation and the maintenance of inflammation at sites of infection. Aberrant neutrophil activity is deleterious; suppressed responses can cause extreme susceptibility to infection while overactivation can lead to excessive inflammation and tissue damage. This review will focus on neutrophil regulation by granulocyte colony-stimulating factor (G-CSF), the principal cytokine controlling neutrophil development and function. The review will emphasize the molecular aspects of G-CSF-driven granulopoiesis in steady state (healthy) conditions and during demand-driven or 'emergency' conditions elicited by infection or clinical administration of G-CSF. Understanding the molecular control of granulopoiesis will aid in the development of new approaches designed to treat disorders of neutrophil production and function.     

Heparin--a unique stimulator of human colon cancer cells' growth

The cancer microenvironment and the interactions between cancer and surrounding tissue cells are thought to play a pivotal role in tumor development and progression. Glycosaminoglycans (GAGs)/proteoglycans (PGs) are major constituents of the extracellular matrix, the composition of which may affect various cellular functions. In the present study, the effects of GAGs on the proliferation of HT29, SW1116, and HCT116 human colon cancer cell lines were examined using exogenously added GAGs, an inhibitor of endogenous GAG sulfation and specific glycosidase digestions. Our results demonstrate that colon cancer cell growth was exclusively stimulated by exogenously added heparin and insensitive to endogenous GAGs/PGs production, in a sulfation pattern-related manner. Treatment of the tested cell lines with the FGF-2 neutralizing antibody showed that the stimulatory effect of heparin on the cells' growth was not FGF-2-dependent. Responsiveness of colon cancer cell lines to exogenous heparin/heparan sulfate may play a role in their growth and metastasis.     

Population structure of an endemic vulnerable species, the Jamaican boa (Epicrates subflavus)

The Jamaican boa (Epicrates subflavus; also called Yellow boa) is an endemic species whose natural populations greatly and constantly declined since the late 19th century, mainly because of predation by introduced species, human persecution, and habitat destruction. In-situ conservation of the Jamaican boa is seriously hindered by the lack of information on demographic and ecological parameters as well as by a poor understanding of the population structure and species distribution in the wild. Here, using nine nuclear microsatellite loci and a fragment of the mitochondrial cytochrome b gene from 87 wild-born individuals, we present the first molecular genetic analyses focusing on the diversity and structure of the natural populations of the Jamaican boa. A model-based clustering analysis of multilocus microsatellite genotypes identifies three groups that are also significantly differentiated on the basis of F-statistics. Similarly, haplotypic network reconstruction methods applied on the cytochrome b haplotypes isolated here identify two well-differentiated haplogroups separated by four to six fixed mutations. Bayesian and metaGA analyses of the mitochondrial data set combined with sequences from other Boidae species indicate that rooting of the haplotypic network occurs most likely between the two defined haplogroups. Both analyses (based on nuclear and mitochondrial markers) underline an Eastern vs. (Western + Central) pattern of differentiation in agreement with geological data and patterns of differentiation uncovered in other vertebrate and invertebrate Jamaican species. Our results provide important insights for improving management of ex-situ captive populations and for guiding the development of proper in-situ species survival and habitat management plans for this spectacular, yet poorly known and vulnerable, snake.     

Myosin regulation of NKCC1: effects on cAMP-mediated Cl- secretion in intestinal epithelia

The basally located actin cytoskeleton has been demonstratedpreviously to regulate Clsecretion from intestinal epithelia via its effects on theNa⁺-K⁺-2Clcotransporter (NKCC1). In nontransporting epithelia, inhibition ofmyosin light chain kinase (MLCK) prevents cell-shrinkage-induced activation of NKCC1. The aim of this study was to investigate the roleof myosin in the regulation of secretagogue-stimulated Cl secretion in intestinalepithelia. The human intestinal epithelial cell line T84 was used forthese studies. Prevention of myosin light chain phosphorylation withthe MLCK inhibitor ML-9 or ML-7 and inhibition of myosin ATPase withbutanedione monoxime (BDM) attenuated cAMP but notCa²⁺-mediatedCl secretion. Both ML-9 andBDM diminished cAMP activation of NKCC1. Neither apicalCl channel activity,basolateral K⁺ channel activity,norNa⁺-K⁺-ATPasewere affected by these agents. Cytochalasin D prevented suchattenuation. cAMP-induced rearrangement of basal actin microfilaments was prevented by both ML-9 and BDM. The phosphorylation of mosin lightchain and subsequent contraction of basal actin-myosin bundles arecrucial to the cAMP-driven activation of NKCC1 and subsequent apicalCl efflux.

     

Mn-induced changes in leaf structure and chloroplast ultrastructure of Citrus volkameriana (L.) plants

Seedlings of Citrus volkameriana (L.) were grown hydroponically for 43 days in order to study the effect of Mn concentration (0, 2, 14, 98 and 686 microM) in the nutrient solution on leaf anatomy and mesophyll chloroplast ultrastructure. Increasing Mn concentration stimulated leaf lamina thickness. The size of mesophyll chloroplasts decreased and increased under 0 and 686 microM Mn, respectively, compared to the intermediate Mn concentrations, similar with regard to the number of chloroplasts per mesophyll cell area. Thylakoid membranes of plants grown under 0 microM Mn were somewhat swelled, while those in other Mn treatments did not present any visible malformation. The relative volume of starch grains per chloroplast was significantly smaller under 0-98 microM Mn (12.8-16.0%) than in the treatment with 686 microM Mn (67.6%). Further, under 686 microM Mn, dark deposits were found in vacuoles. The existence of a cell adaptation mechanism to excessive Mn availability (686 microM Mn) by increasing the size of chloroplasts as well as their number per cellular area, is discussed.     

The acid adaptive tolerance response in Campylobacter jejuni induces a global response,as suggested by proteomics and microarrays

Campylobacter jejuni CI 120 is a natural isolate obtained during poultry processing and has the ability to induce an acid tolerance response (ATR) to acid + aerobic conditions in early stationary phase. Other strains tested they did not induce an ATR or they induced it in exponential phase. Campylobacter spp. do not contain the genes that encode the global stationary phase stress response mechanism. Therefore, the aim of this study was to identify genes that are involved in the C. jejuni CI 120 early stationary phase ATR, as it seems to be expressing a novel mechanism of stress tolerance. Two-dimensional gel electrophoresis was used to examine the expression profile of cytosolic proteins during the C. jejuni CI 120 adaptation to acid + aerobic stress and microarrays to determine the genes that participate in the ATR. The results indicate induction of a global response that activated a number of stress responses, including several genes encoding surface components and genes involved with iron uptake. The findings of this study provide new insights into stress tolerance of C. jejuni, contribute to a better knowledge of the physiology of this bacterium and highlight the diversity among different strains.     

Synthesis and Evaluation of Chloramphenicol Homodimers: Molecular Target,Antimicrobial Activity,and Toxicity against Human Cells

As fight against antibiotic resistance must be strengthened, improving old drugs that have fallen in reduced clinical use because of toxic side effects and/or frequently reported resistance, like chloramphenicol (CAM), is of special interest. Chloramphenicol (CAM), a prototypical wide-spectrum antibiotic has been shown to obstruct protein synthesis via binding to the bacterial ribosome. In this study we sought to identify features intensifying the bacteriostatic action of CAM. Accordingly, we synthesized a series of CAM-dimers with various linker lengths and functionalities and compared their efficiency in inhibiting peptide-bond formation in an Escherichia coli cell-free system. Several CAM-dimers exhibited higher activity, when compared to CAM. The most potent of them, compound 5, containing two CAM bases conjugated via a dicarboxyl aromatic linker of six successive carbon-bonds, was found to simultaneously bind both the ribosomal catalytic center and the exit-tunnel, thus revealing a second, kinetically cryptic binding site for CAM. Compared to CAM, compound 5 exhibited comparable antibacterial activity against MRSA or wild-type strains of Staphylococcus aureus, Enterococcus faecium and E. coli, but intriguingly superior activity against some CAM-resistant E. coli and Pseudomonas aeruginosa strains. Furthermore, it was almost twice as active in inhibiting the growth of T-leukemic cells, without affecting the viability of normal human lymphocytes. The observed effects were rationalized by footprinting tests, crosslinking analysis, and MD-simulations.     

Relationships Between the Properties of Self-Emulsifying Pellets and of the Emulsions Used as Massing Liquids for Their Preparation

Self-emulsifying pellets were prepared using microcrystalline cellulose, emulsions of caprylic/capric triglyceride, and three Cremophors (ELP, RH40, and RH60) at 1.5 and 2.3 weight ratios, and two drugs (furosemide and propranolol) of different lipophilicity. Droplet size, zeta potential (ζ) and viscosity of emulsions, and pellet size, shape, friability, tensile strength, disintegration, and drug migration in pellets were determined. Evaluation of reconstituted emulsions was based on droplet size and ζ. Factorial design and 3-way ANOVA was applied to estimate the significance of the effects of the drug, surfactant and oil/surfactant ratio. It was found that droplet size, viscosity and ζ of emulsions, and size, shape, and friability of pellets were affected by the studied factors and were significant interactions between their effects on pellet size and friability. Migration of drug towards the pellet surface was higher for the less lipophilic furosemide and higher oil content. Linear relationships were found between the emulsion viscosity and the shape parameters of the pellets (for the aspect ratio R² = 0.796 for furosemide and R² = 0.885 for propranolol and for the shape factor, e_RR² = 0.740 and R² = 0.960, respectively). For all the formulations examined, an exponential relationship was found between migration (M%) and the product of viscosity (η) and solubility of drug in oil/surfactant mixture (S) (M% = 98.1e-0.016 [η•S], R² = 0.856), which may be useful in formulation work.KEY WORDS: drug distribution, emulsion and pellet characterization, friability and tensile strength, furosemide and propranolol, self-emulsifying pellets     

The metabolome of induced pluripotent stem cells reveals metabolic changes occurring in somatic cell reprogramming

Metabolism is vital to every aspect of cell function, yet the metabolome of induced pluripotent stem cells (iPSCs) remains largely unexplored. Here we report, using an untargeted metabolomics approach, that human iPSCs share a pluripotent metabolomic signature with embryonic stem cells (ESCs) that is distinct from their parental cells, and that is characterized by changes in metabolites involved in cellular respiration. Examination of cellular bioenergetics corroborated with our metabolomic analysis, and demonstrated that somatic cells convert from an oxidative state to a glycolytic state in pluripotency. Interestingly, the bioenergetics of various somatic cells correlated with their reprogramming efficiencies. We further identified metabolites that differ between iPSCs and ESCs, which revealed novel metabolic pathways that play a critical role in regulating somatic cell reprogramming. Our findings are the first to globally analyze the metabolome of iPSCs, and provide mechanistic insight into a new layer of regulation involved in inducing pluripotency, and in evaluating iPSC and ESC equivalence.