Lipolysis - a highly regulated multi-enzyme complex mediates the catabolism of cellular fat stores

Lipolysis is the biochemical pathway responsible for the catabolism of triacylglycerol (TAG) stored in cellular lipid droplets. The hydrolytic cleavage of TAG generates non-esterified fatty acids, which are subsequently used as energy substrates, essential precursors for lipid and membrane synthesis, or mediators in cell signaling processes. Consistent with its central importance in lipid and energy homeostasis, lipolysis occurs in essentially all tissues and cell types, it is most abundant, however, in white and brown adipose tissue. Over the last 5years, important enzymes and regulatory protein factors involved in lipolysis have been identified. These include an essential TAG hydrolase named adipose triglyceride lipase (ATGL) [annotated as patatin-like phospholipase domain-containing protein A2], the ATGL activator comparative gene identification-58 [annotated as α/β hydrolase containing protein 5], and the ATGL inhibitor G0/G1 switch gene 2. Together with the established hormone-sensitive lipase [annotated as lipase E] and monoglyceride lipase, these proteins constitute the basic "lipolytic machinery". Additionally, a large number of hormonal signaling pathways and lipid droplet-associated protein factors regulate substrate access and the activity of the "lipolysome". This review summarizes the current knowledge concerning the enzymes and regulatory processes governing lipolysis of fat stores in adipose and non-adipose tissues. Special emphasis will be given to ATGL, its regulation, and physiological function.     

Movements of the epsilon-subunit during catalysis and activation in single membrane-bound H(+)-ATP synthase

F0F1-ATP synthases catalyze proton transport-coupled ATP synthesis in bacteria, chloroplasts, and mitochondria. In these complexes, the epsilon-subunit is involved in the catalytic reaction and the activation of the enzyme. Fluorescence-labeled F0F1 from Escherichia coli was incorporated into liposomes. Single-molecule fluorescence resonance energy transfer (FRET) revealed that the epsilon-subunit rotates stepwise showing three distinct distances to the b-subunits in the peripheral stalk. Rotation occurred in opposite directions during ATP synthesis and hydrolysis. Analysis of the dwell times of each FRET state revealed different reactivities of the three catalytic sites that depended on the relative orientation of epsilon during rotation. Proton transport through the enzyme in the absence of nucleotides led to conformational changes of epsilon. When the enzyme was inactive (i.e. in the absence of substrates or without membrane energization), three distances were found again, which differed from those of the active enzyme. The three states of the inactive enzyme were unequally populated. We conclude that the active-inactive transition was associated with a conformational change of epsilon within the central stalk.     

Detailed Comparative Map of Human Chromosome 19q and Related Regions of the Mouse Genome

One of the larger contiguous blocks of mouse–human genomic homology includes the proximal portion of mouse chromosome 7 and the long arm of human chromosome 19. Previous studies have demonstrated the close relationship between the two regions, but have also indicated significant rearrangements in the relative orders of homologous mouse and human genes. Here we present the genetic locations of the homologs of 42 human chromosome 19q markers in the mouse, with an emphasis on genes also included in the human chromosome 19 physical map. Our results demonstrate that despite an overall inversion of sequences relative to the centromere, apparent “transpositions” of three gene-rich segments, and a local inversion of markers mapping near the 19q telomere, gene content, order, and spacing are remarkably well conserved throughout the lengths of these related mouse and human regions. Although most human 19q markers have remained genetically linked in mouse, one small human segment forms a separate region of homology between human chromosome 19q and mouse chromosome 17. Three of the four rearrangements of mouse versus human 19q sequences involve segments that are located directly adjacent to each other in 19q13.3–q13.4, suggesting either the coincident occurrence of these events or their common association with unstable DNA sequences. These data permit an unusually in-depth examination of this large region of mouse–human genomic homology and provide an important new tool to aid in the mapping of genes and associated phenotypes in both species.     

The immunogenicity of dendritic cell-based vaccines is not hampered by doxorubicin and melphalan administration

Immunization of cancer patients is most effective in tumor-free conditions or in the presence of minimal residual disease. In the attempt to develop new strategies able to control tumor recurrence while allowing the development of protective immunity, we have investigated the immunogenic potential of two distinct vaccine formulations when provided alone or upon single and repeated treatment with chemotherapeutics drugs. Vaccine-induced T cell responses were first investigated by tracing Ag-specific T cell responses in mice bearing detectable frequencies of Ag-specific TCR transgenic CD4 and CD8 T cells. These studies indicated that immunization with peptide-pulsed dendritic cells and soluble Ag plus adjuvant elicited a comparable expansion and differentiation of CD4 and CD8 effector cells in the peripheral lymphoid tissues when provided alone or shortly after Doxorubicin or Melphalan administration. We also analyzed the potency of the combined vaccination in transgenic adenocarcinoma mouse prostate mice, which develop spontaneous prostate cancer. Dendritic cell-based vaccination elicited potent tumor-specific cytotoxic responses in mice bearing prostate intraepithelial neoplasia both in the absence and in the presence of Doxorubicin. Together our results indicate that Doxorubicin- or Melphalan-based chemotherapy and Ag-specific vaccination can be combined for adjuvant treatments of cancer patients.     

Horizontal Transmission of the Entomopathogenic Fungus Beauveria bassiana among the Spruce Bark Beetle, Ips typographus (Col., Scolytidae) in the Laboratory and under Field Conditions

In laboratory and field cage experiments, the horizontal transmission of the entomopathogenic fungus Beauveria bassiana (Boverol®) between adults of the spruce bark beetle Ips typographus was examined. In a laboratory experiment, conidia transfer between treated and untreated beetles at different ratios resulted in 96% mortality (MST 4.3 days) at a ratio of 1:1 and 90% mortality (MST 4.5 days) at a ratio of 1:2. At ratios of 1:5, 1:10 and 1:20, the mortalities at 7 days were 83, 77 and 75%, respectively. In a second laboratory experiment, the efficacy of B. bassiana was investigated after transmission from contaminated to healthy beetles over a period of 5 days. Two weeks later, the mortality of treated and untreated beetles was 99% (control mortality was 44%), while the rate of mycosis was 79% in the treatment compared to 10% in the control beetles. A single contact between one treated male (2.0×10⁵ conidia/beetle) and an untreated female was found to be sufficient to transmit a lethal dose of B. bassiana of about 1.2×10⁴ conidia. Two experiments in field cages in a spruce stand demonstrated horizontal transmission of B. bassiana (Boverol®) between beetles. In the first experiment the transmission was investigated following the introduction of B. bassiana-inoculated beetles into a population of untreated beetles. Significant reductions were observed in the length of maternal galleries, and the number of larvae and pupae. In the second field cage experiment, natural populations of beetles were lured into a flight cage using a pheromone trap, and treated with B. bassiana or left untreated on alternate days. In this experiment, significant reductions were observed in the number of bore holes and the length of maternal galleries, while no larvae, pupae or juveniles were found under the bark of spruce trunks in the Boverol® transmission treatment. It is concluded that B. bassiana has potential for control of I. typographus, and further field experiments are warranted to investigate its efficacy under more practical conditions.     

Cross-Scale Responses of Biodiversity to Hurricane and Anthropogenic Disturbance in a Tropical Forest

Abstract In studies of biodiversity, considerations of scale—the spatial or temporal domain to which data provide inference—are important because of the non-arithmetic manner in which species richness increases with area (and total abundance) and because fine-scale mechanisms (for example, recruitment, growth, and mortality of species) can interact with broad scale patterns (for example, habitat patch configuration) to influence dynamics in space and time. The key to understanding these dynamics is to consider patterns of environmental heterogeneity, including patterns produced by natural and anthropogenic disturbance. We studied how spatial variation in three aspects of biodiversity of terrestrial gastropods (species richness, species diversity, and nestedness) on the 16-ha Luquillo Forest Dynamics Plot (LFDP) in a tropical forest of Puerto Rico was affected by disturbance caused by Hurricanes Hugo and Georges, as well as by patterns of historic land use. Hurricane-induced changes in spatial organization of species richness differed from those for species diversity. The gamma components of species richness changed after the hurricanes and were significantly different between Hurricanes Hugo and Georges. Alpha and two beta components of species richness, one related to turnover among sites within areas of similar land use and one related to variation among areas of different land use, varied randomly over time after both hurricanes. In contrast, gamma components of species diversity decreased in indistinguishable manners after both hurricanes, whereas the rates of change in the alpha component of species diversity differed between hurricanes. Beta components of diversity related to turnover among sites declined after both hurricanes in a consistent fashion. Those related to turnover among areas with different historic land uses varied stochastically. The immediate effect of hurricanes was to reduce nestedness of gastropod assemblages. Thereafter, nestedness increased during post-hurricane secondary succession, and did so in the same way, regardless of patterns of historic land use. The rates of change in degree of nestedness during secondary succession were different after each hurricane as a result of differences in the severity and extent of the hurricane-induced damage. Our analyses quantified temporal changes in the spatial organization of biodiversity of gastropod assemblages during forest recovery from hurricane-induced damage in areas that had experienced different patterns of historic human land use, and documented the dependence of biodiversity on spatial scale. We hypothesize that cross-scale interactions, likely those between the local demographics of species at the fine scale and the landscape configuration of patches at the broad scale, play a dominant role in affecting critical transfer processes, such as dispersal, and its interrelationship with aspects of biodiversity. Cross-scale interactions have significant implications for the conservation of biodiversity, as the greatest threats to biodiversity arise from habitat modification and fragmentation associated with disturbance arising from human activities.     

Computational genes: a tool for molecular diagnosis and therapy of aberrant mutational phenotype

Background  

A finite state machine manipulating information-carrying DNA strands can be used to perform autonomous molecular-scale computations at the cellular level.     

Metal-protein complex-mediated transport and delivery of Ni2+ to TCR/MHC contact sites in nickel-specific human T cell activation

Nickel allergy clearly involves the activation of HLA-restricted, skin-homing, Ni-specific T cells by professional APCs. Nevertheless, knowledge concerning the molecular details of metal-protein interactions underlying the transport and delivery of metal ions to APC during the early sensitization phase and their interactions with HLA and TCRs is still fragmentary. This study investigates the role of human serum albumin (HSA), a known shuttling molecule for Ni(2+) and an often-disregarded, major component of skin, in these processes. We show that Ni-saturated HSA complexes (HSA-Ni) induce and activate Ni-specific human T cells as potently as Ni salt solutions when present at equimolar concentrations classically used for in vitro T cell stimulation. However, neither HSA itself nor its Ni-binding N-terminal peptide are involved in determining the specificity of antigenic determinants. In fact, HSA could be replaced by xenogeneic albumins exhibiting sufficient affinity for Ni(2+) as determined by surface plasmon resonance (Biacore technology) or atomic absorption spectroscopy. Moreover, despite rapid internalization of HSA-Ni by APC, it was not processed into HLA-associated epitopes recognizable by Ni-specific T cells. In contrast, the presence of HSA-Ni in the vicinity of transient contacts between TCR and APC-exposed HLA molecules appeared to facilitate a specific transfer of Ni(2+) from HSA to high-affinity coordination sites created at the TCR/HLA-interface.     

Intracellular Delivery of Carbohydrates into Mammalian Cells through Swelling-activated Pathways

Volume changes of human T-lymphocytes (Jurkat line) exposed to hypotonic carbohydrate-substituted solutions of different composition and osmolality were studied by videomicroscopy. In 200 mOsm media the cells first swelled within 1–2 min and then underwent regulatory volume decrease (RVD) to their original isotonic volume within 10–15 min. RVD also occurred in strongly hypotonic 100 mOsm solutions of di- and trisaccharides (trehalose, sucrose, raffinose). In contrast to oligosaccharide media, 100 mOsm solutions of monomeric carbohydrates (glucose, galactose, inositol and sorbitol) inhibited RVD. The complex volumetric data were analyzed with a membrane transport model that allowed the estimation of the hydraulic conductivity and volume-dependent solute permeabilities. We found that under slightly hypotonic stress (200 mOsm) the cell membrane was impermeable to all carbohydrates studied here. Upon osmolality decrease to 100 mOsm, the membrane permeability to monomeric carbohydrates increased dramatically (apparently due to channel activation caused by extensive cell swelling), whereas oligosaccharide permeability remained very poor. The size-selectivity of the swelling-activated sugar permeation was confirmed by direct chromatographic measurements of intracellular sugars. The results of this study are of interest for biotechnology, where sugars and related compounds are increasingly being used as potential cryo- and lyoprotective agents for preservation of rare and valuable mammalian cells and tissues.This revised version was published online in June 2005 with a corrected cover date.