Toroidal nucleoids in Escherichia coli exposed to chloramphenicol

The DNA of growing cells of Escherichia coli occurs in one or a few lobular bodies known as nucleoids. Upon exposure to chloramphenicol, the nucleoids assume compact, rounded forms ("cm-nucleoids") that have been described as ring- or sphere-shaped. Multiple views of single cells or spheroplasts, however, support a different, curved toroid shape for cm-nucleoids. The multiple views were obtained either by DNA fluorescence imaging as the cells or spheroplasts reoriented in liquid medium or by optical sectioning using phase-contrast or fluorescence imaging of immobilized cells. The curved toroid shape is consistent with electron microscope images of thin sections of chloramphenicol-treated cells. The relationship of this structure to active and inactive nucleoids and to the smaller toroidal forms made by in vitro DNA condensation is discussed.     

Genetic screen for small body size mutants in C. elegans reveals many TGFbeta pathway components

In the nematode Caenorhabditis elegans, a TGFbeta-related signaling pathway regulates body size and male tail morphogenesis. We sought to identify genes encoding components or modifiers of this pathway in a large-scale genetic screen. Remarkably, this screen was able to identify essentially all core components of the TGFbeta signaling pathway. Among 34 Small mutants, many mutations disrupt genes encoding recognizable components of the TGFbeta pathway: DBL-1 ligand, DAF-4 type II receptor, SMA-6 type I receptor, and SMA-2, SMA-3, and SMA-4 Smads. Moreover, we find that at least 11 additional complementation groups can mutate to the Small phenotype. Four of these 11 genes, sma-9, sma-14, sma-16, and sma-20 affect male tail morphogenesis as well as body size. Two genes, sma-11 and sma-20, also influence regulation of the developmentally arrested dauer larval stage, suggesting a role in a second characterized TGFbeta pathway in C. elegans. Other genes may represent tissue-specific factors or parallel pathways for body size control. Because of the conservation of TGFbeta signaling pathways, homologs of these genes may be involved in tissue specificity and/or crosstalk of TGFbeta pathways in other animals.     

Upregulation of vascular endothelial growth factor (VEGF) in the retinas of transgenic mice overexpressing interleukin-1beta (IL-1beta) in the lens and mice undergoing retinal degeneration

IL-1beta is a pro-inflammatory agent associated with angiogenesis and increased vascular permeability. To determine whether IL-1beta elicits these responses through an upregulation of VEGF, transgenic mice that overexpress IL-1beta in the lens were evaluated at various time points for the localization of VEGF, the location and extent of blood-retinal barrier (BRB) breakdown, and the origin and extent of neovascularization (NV). In homozygous and heterozygous transgenic mice, but not controls, intense VEGF immunoreactivity was scattered throughout the retina at postnatal days 5-7 (P5-7), just after the onset of inflammatory cell infiltration. VEGF staining in the retina remained widespread, but weak from P9-15. Beginning at P15, the intensity of VEGF immunoreactivity achieved a second peak, which it maintained through adulthood. This peak coincided with significant retinal destruction due to massive inflammation. The onset of BRB breakdown coincided with the upregulation of VEGF (P5-7) and widespread BRB breakdown was demonstrated from about P9. From P9-12, aggregates of cells positive for Griffonia simplicifolia isolectin-B4, a marker for vascular endothelial cells, formed on the retinal surface. These cells migrated into the retina at P12-15 with the more superficial cells forming a network of vessels and the deeper cells remaining in small clusters, thus demonstrating that NV occurs much later than BRB breakdown. Non-transgenic FVB/N mice, which undergo retinal degeneration beginning at about P9, also demonstrate the latter peak of VEGF upregulation and the accompanying BRB breakdown, but not the early upregulation. VEGF immunostaining of transgenic and non-transgenic mouse retinas was eliminated by pre-incubation of the VEGF antibodies with VEGF peptide. The data suggest that the early peak of VEGF upregulation (P5-7) and its accompanying BRB breakdown is due to IL-1beta expression and is likely to be dependent on inflammatory cell infiltration. The latter peak appears to be related to retinal destruction.     

Cytometric quantification of nitrate reductase by immunolabeling in the marine diatom Skeletonema costatum

BACKGROUND: The uptake of nitrate by phytoplankton is a central issue in biological oceanography due to its importance to primary production and vertical flux of biogenic carbon. Nitrate reductase catalyzes the first step of nitrate assimilation, the reduction of NO(3) to NO(2). A cytometric protocol to detect and quantify relative changes in nitrate reductase (NR) protein content of the marine centric diatom Skeletonema costatum is presented. METHODS: Immunolabeling of NR protein was achieved with polyclonal antibodies raised against S.costatum NR. Antisera specific to a NR protein subunit and to a NR polypeptide sequence were compared, and cytometric results of NR protein abundance were related to Western analyses. Changes in cellular NR abundance and activity were followed during an upwelling simulation experiment in which S. costatum was exposed to a shift from ammonia to nitrate as major nitrogen source. RESULTS: NR protein could be detected in NO(3)-grown cells and at extremely low levels hardly discernible by Western Blot densiometry in NH(4)-grown cells. The protocol allowed observation of early stages of NR induction during an upwelling simulation. NR abundance increased after the nutrient shift to reach a new physiological "steady-state" 96 hrs later. NR activity exhibited diel variation with maxima at mid-day. NR abundance as estimated by both flow cytometry and Western analysis exhibited a hyperbolic relationship to NR activity. This pattern suggests post-translational activation of NR protein. CONCLUSIONS: The presented protocol allows the differentiation of NH(4)- versus NO(3)-grown algae as well as the monitoring of early stages in the induction of nitrate assimilatory capacities.     

Fatty acid binding proteins from different tissues show distinct patterns of fatty acid interactions

Fatty acid binding proteins (FABP) form a family of proteins displaying tissue-specific expression. These proteins are involved in fatty acid (FA) transport and metabolism by mechanisms that also appear to be tissue-specific. Cellular retinoid binding proteins are related proteins with unknown roles in FA transport and metabolism. To better understand the origin of these tissue-specific differences we report new measurements, using the acrylodated intestinal fatty acid binding protein (ADIFAB) method, of the binding of fatty acids (FA) to human fatty acid binding proteins (FABP) from brain, heart, intestine, liver, and myelin. We also measured binding of FA to a retinoic acid (CRABP-I) and a retinol (CRBP-II) binding protein and we have extended to 19 different FA our characterization of the FA-ADIFAB and FA-rat intestinal FABP interactions. These studies extend our previous analyses of human FABP from adipocyte and rat FABPs from heart, intestine, and liver. Binding affinities varied according to the order brain approximately myelin approximately heart > liver > intestine > CRABP > CRBP. In contrast to previous studies, no protein revealed a high degree of selectivity for particular FA. The results indicate that FA solubility (hydrophobicity) plays a major role in governing binding affinities; affinities tend to increase with increasing hydrophobicity (decreasing solubility) of the FA. However, our results also reveal that, with the exception of the intestinal protein, FABPs exhibit an additional attractive interaction for unsaturated FA that partially compensates for their trend toward lower affinities due to their higher aqueous solubilities. Thermodynamic potentials were determined for oleate and arachidonate binding to a subset of the FABP and retinoid binding proteins. FA binding to all FABPs was enthalpically driven. The DeltaH degrees values for paralogous FABPs, proteins from the same species but different tissues, reveal an exceptionally wide range of values, from -22 kcal/mol (myelin) to -7 kcal/mol (adipocyte). For orthologous FABPs from the same tissue but different species, DeltaH degrees values were similar. In contrast to the enthalpic dominance of FA binding to FABP, binding of FA to CRABP-I was entropically driven. This is consistent with the notion that FA specificity for FABP is determined by the enthalpy of binding. Proteins from different tissues also revealed considerable heterogeneity in heat capacity changes upon FA binding, DeltaC(p) values ranged between 0 and -1.3 kcal mol(-1) K(-1). The results demonstrate that thermodynamic parameters are quite different for paralogous but are quite similar for orthologous FABP, suggesting tissue-specific differences in FABP function that may be conserved across species.     

The calpain small subunit gene is essential: its inactivation results in embryonic lethality

Creation of transgenic (knockout) mice deficient in calpain small (30 kDa) subunit gene was undertaken to clarify the proposed role of the small subunit for calpain proteolytic activity and to gain insight into the importance of the gene in the whole animal. The gene was targeted and disrupted in embryonic stem cells by homologous recombination, and chimeric mice were generated. Heterozygous F1 generation mice were crossed to obtain F2 generation. Among F2 generation mice, we found only wild-type and heterozygous animals in the 80 pups genotyped to date; no homozygous mice have been found, although 20 were expected. The heterozygotes had no apparent phenotypic abnormalities. Analysis of their tissues revealed no significant difference in mRNA expression, protein content, or proteolytic activity in comparison with their wild-type littermates. Genotyping of fetuses at different stages of development also revealed only wild-type and normal heterozygous fetuses. No moribund embryos or resorption sites were observed in the uterine cavity. The results indicate that at least one normal allele is essential for postnatal survival. Disruption of both alleles appears to be lethal in very early fetal development.     

Expression of fatty acid-CoA ligase 4 during development and in brain

Fatty acid utilization is initiated by fatty acid-CoA ligase, which converts free fatty acids into fatty acyl-CoA esters. We have cloned previously the human long-chain fatty acid-CoA ligase 4 (FACL4), which is a central enzyme in controlling the free arachidonic acid level in cells and thereby regulating eicosanoid production. We report here the expression of this gene in tissues, particularly in different parts of the brain. We found that FACL4 encoded a 75 kDa enzyme and that there was a modified translation product expressed in the brain. FACL4 was expressed in early stages of development with a significant amount of FACL4 mRNA detected in an E7 mouse embryo. In addition, FACL4 was highly expressed in both adult and newborn mouse brain especially in the granule cells of the dentate gyrus and the pyramidal cell layer of CA1 in hippocampus, and the granular cell layer and Purkinje cells of the cerebellum.     

Mitotic catastrophe triggered in human cancer cells by the viral protein apoptin

Mitotic catastrophe is an oncosuppressive mechanism that senses mitotic failure leading to cell death or senescence. As such, it protects against aneuploidy and genetic instability, and its induction in cancer cells by exogenous agents is currently seen as a promising therapeutic end point. Apoptin, a small protein from Chicken Anemia Virus (CAV), is known for its ability to selectively induce cell death in human tumor cells. Here, we show that apoptin triggers p53-independent abnormal spindle formation in osteosarcoma cells. Approximately 50% of apoptin-positive cells displayed non-bipolar spindles, a 10-fold increase as compared to control cells. Besides, tumor cells expressing apoptin are greatly limited in their progress through anaphase and telophase, and a significant drop in mitotic cells past the meta-to-anaphase transition is observed. Time-lapse microscopy showed that mitotic osteosarcoma cells expressing apoptin displayed aberrant mitotic figures and/or had a prolonged cycling time during mitosis. Importantly, all dividing cells expressing apoptin eventually underwent cell death either during mitosis or during the following interphase. We infer that apoptin can efficiently trigger cell death in dividing human tumor cells through induction of mitotic catastrophe. However, the killing activity of apoptin is not only confined to dividing cells, as the CAV-derived protein is also able to trigger caspase-3 activation and apoptosis in non-mitotic cancer cells.