An integrated physical and genetic map of the nematode<Emphasis Type="Italic"> Pristionchus pacificus</Emphasis>

The free-living nematode Pristionchus pacificus is one of several species that have recently been developed as a satellite system for comparative functional studies in evolutionary developmental biology. Comparisons of developmental processes between P. pacificus and the well established model organism Caenorhabditis elegans at the cellular and genetic levels provide detailed insight into the molecular changes that shape evolutionary transitions. To facilitate genetic analysis and cloning of mutations in P. pacificus, we previously generated a BAC-based genetic linkage map for this organism. Here, we describe the construction of a physical map of the P. pacificus genome based on AFLP fingerprint analysis of 7747 BAC clones. Most of the SSCP markers used to generate the genetic linkage map were derived from BAC ends, so that the physical genome map and the genetic map can be integrated. The contigs that make up the physical map are evenly distributed over the genetic linkage map and no clustering is observed, indicating that the physical map provides a valid representation of the P. pacificus genome. The integrated genome map thus provides a framework for positional cloning and the study of genome evolution in nematodes.Communicated by G. Jürgens     

Effects of the Ca ionophore A23187 on zinc-induced apoptosis in C6 glioma cells

Zinc ions are essential, but at elevated concentrations, they also have toxic effects on mammalian cells. Zinc plays a crucial role in cell proliferation and differentiation and it even protects cells against apoptosis caused by various reagents. On the other hand, zinc at high concentrations causes cell death that was characterized as apoptotic by internucleosomal DNA fragmentation, formation of apoptotic bodies, and breakdown of the mitochondrial membrane potential. In the present work, a clone of rat C6 glioma cells that was resistant to toxic effects of ZnCl₂ up to 250 μM was employed to study the effect of the ionophore A23187 on zinc-induced apoptosis. Neither 150 μM Zn²⁺ nor 100 nM A23187 alone caused apoptosis as measured by internucleosomal DNA fragmentation. However, combined exposure of C6 cells to 100 nM A23187 and 150 μM Zn²⁺ for 48 h was effective in inducing apoptosis. Because the so-called calcium ionophore A23187 is not specific for Ca²⁺ ions but also transports Zn²⁺ with high selectivity over Ca²⁺, we investigated whether this substance promoted the uptake of Zn²⁺ ions into C6 cells. Employing the zinc-specific fluorescence probe Zinquin, we observed that the very low concentration of 1.9 nM A23187 significantly and rapidly raised the intracellular mobile Zn²⁺ content. Analysis by atomic absorption spectroscopy revealed that incubation with 1.9 nM A23187 caused a doubling of the total intracellular zinc level within 60 min. We conclude that the apoptosis evoked by the combined action of Zn²⁺ and A23187 was the result of enhanced Zn²⁺ influx evoked by the ionophore, resulting in higher intracellular zinc levels.     

Role of increased guanosine triphosphate cyclohydrolase-1 expression and tetrahydrobiopterin levels upon T cell activation

Tetrahydrobiopterin (BH(4)) is an essential co-factor for the nitric-oxide (NO) synthases, and in its absence these enzymes produce superoxide (O(2)(·-)) rather than NO. The rate-limiting enzyme for BH(4) production is guanosine triphosphate cyclohydrolase-1 (GTPCH-1). Because endogenously produced NO affects T cell function, we sought to determine whether antigen stimulation affected T cell GTPCH-1 expression and ultimately BH(4) levels. Resting T cells had minimal expression of inducible NOS (NOS2), endothelial NOS (NOS3), and GTPCH-1 protein and nearly undetectable levels of BH(4). Anti-CD3 stimulation of T cells robustly stimulated the coordinated expression of NOS2, NOS3, and GTPCH-1 and markedly increased both GTPCH-1 activity and T cell BH(4) levels. The newly expressed GTPCH-1 was phosphorylated on serine 72 and pharmacological inhibition of casein kinase II reduced GTPCH-1 phosphorylation and blunted the increase in T cell BH(4). Inhibition of GTPCH-1 with diaminohydroxypyrimidine (1 mmol/liter) prevented T cell BH(4) accumulation, reduced NO production, and increased T cell O(2)(·-) production, due to both NOS2 and NOS3 uncoupling. GTPCH-1 inhibition also promoted TH(2) polarization in memory CD4 cells. Ovalbumin immunization of mice transgenic for an ovalbumin receptor (OT-II mice) confirmed a marked increase in T cell BH(4) in vivo. These studies identify a previously unidentified consequence of T cell activation, promoting BH(4) levels, NO production, and modulating T cell cytokine production.     

The genes coding for histone H3 and H4 in Neurospora crassa are unique and contain intervening sequences.

Sequences coding for histone H3 and H4 of Neurospora crassa could be identified in genomic digests with the use of the corresponding genes from sea urchin and X. laevis as hybridization probes. A 2.6 kb HindIII-generated N. crassa DNA fragment, showing homology with the heterologous histone H3-gene probes was cloned in a charon 21A vector. Using DNA from this clone as a homologous hybridization probe a 6.9 kb SalI-generated DNA fragment was isolated which in addition to the histone H3-gene also contains the gene coding for histone H4. Several lines of evidence demonstrate the presence of only a single histone H3- as well as a single histone H4-gene in N. crassa. The two genes are physically linked on the genome. DNA sequencing of the N. crassa histone H3- and H4-genes confirmed their identity and, in addition, revealed the presence of one short intron (67 bp) within the coding sequence of the H3-gene and even two introns (68 and 69 bp) within the H4-gene. The amino acid sequences of the N. crassa histones H3 and H4, as deduced from the DNA sequences, and those of the corresponding yeast histones differ only at a few positions. Much larger sequence differences, however, are observed at the DNA level, reflecting a diverging codon usage in the two lower eukaryotes.     

The L1 major capsid protein of human papillomavirus type 11 recombinant virus-like particles interacts with heparin and cell-surface glycosaminoglycans on human keratinocytes

The L1 major capsid protein of human papillomavirus (HPV) type 11, a 55-kDa polypeptide, forms particulate structures resembling native virus with an average particle diameter of 50-60 nm when expressed in the yeast Saccharomyces cerevisiae. We show in this report that these virus-like particles (VLPs) interact with heparin and with cell-surface glycosaminoglycans (GAGs) resembling heparin on keratinocytes and Chinese hamster ovary cells. The binding of VLPs to heparin is shown to exhibit an affinity comparable to that of other identified heparin-binding proteins. Immobilized heparin chromatography and surface plasmon resonance were used to show that this interaction can be specifically inhibited by free heparin and dextran sulfate and that the effectiveness of the inhibitor is related to its molecular weight and charge density. Sequence comparison of nine human L1 types revealed a conserved region of the carboxyl terminus containing clustered basic amino acids that bear resemblance to proposed heparin-binding motifs in unrelated proteins. Specific enzymatic cleavage of this region eliminated binding to both immobilized heparin and human keratinocyte (HaCaT) cells. Removal of heparan sulfate GAGs on keratinocytes by treatment with heparinase or heparitinase resulted in an 80-90% reduction of VLP binding, whereas treatment of cells with laminin, a substrate for alpha6 integrin receptors, provided minimal inhibition. Cells treated with chlorate or substituted beta-D-xylosides, resulting in undersulfation or secretion of GAG chains, also showed a reduced affinity for VLPs. Similarly, binding of VLPs to a Chinese hamster ovary cell mutant deficient in GAG synthesis was shown to be only 10% that observed for wild type cells. This report establishes for the first time that the carboxyl-terminal portion of HPV L1 interacts with heparin, and that this region appears to be crucial for interaction with the cell surface.     

Phylogenetic and evolutionary implications of complete chloroplast genome sequences of four early-diverging angiosperms: Buxus (Buxaceae), Chloranthus (Chloranthaceae), Dioscorea (Dioscoreaceae), and Illicium (Schisandraceae)

We have determined the complete chloroplast genome sequences of four early-diverging lineages of angiosperms, Buxus (Buxaceae), Chloranthus (Chloranthaceae), Dioscorea (Dioscoreaceae), and Illicium (Schisandraceae), to examine the organization and evolution of plastid genomes and to estimate phylogenetic relationships among angiosperms. For the most part, the organization of these plastid genomes is quite similar to the ancestral angiosperm plastid genome with a few notable exceptions. Dioscorea has lost one protein-coding gene, rps16; this gene loss has also happened independently in four other land plant lineages, liverworts, conifers, Populus, and legumes. There has also been a small expansion of the inverted repeat (IR) in Dioscorea that has duplicated trnH-GUG. This event has also occurred multiple times in angiosperms, including in monocots, and in the two basal angiosperms Nuphar and Drimys. The Illicium chloroplast genome is unusual by having a 10 kb contraction of the IR. The four taxa sequenced represent key groups in resolving phylogenetic relationships among angiosperms. Illicium is one of the basal angiosperms in the Austrobaileyales, Chloranthus (Chloranthales) remains unplaced in angiosperm classifications, and Buxus and Dioscorea are early-diverging eudicots and monocots, respectively. We have used sequences for 61 shared protein-coding genes from these four genomes and combined them with sequences from 35 other genomes to estimate phylogenetic relationships using parsimony, likelihood, and Bayesian methods. There is strong congruence among the trees generated by the three methods, and most nodes have high levels of support. The results indicate that Amborella alone is sister to the remaining angiosperms; the Nymphaeales represent the next-diverging clade followed by Illicium; Chloranthus is sister to the magnoliids and together this group is sister to a large clade that includes eudicots and monocots; and Dioscorea represents an early-diverging lineage of monocots just internal to Acorus.     

Human hepatoma (Hep G2) cultures contain salt-resistant triglyceridase ("liver lipase")

The culture fluid of Hep G2 human hepatoma cells contains triglyceridase activity resistant to high-salt concentrations. The lipase binds to Sepharose-heparin columns from which it can be eluted by 0.8 to 0.9 M NaCl. The nature of this lipase was studied using antibodies raised against "liver" lipases from human and rat origin. The anti-rat liver lipase inhibits both the postheparin human and rat plasma enzyme while the anti-human liver lipase has no effect on the rat enzyme. The lipase of the Hep G2 cultures showed affinity to the antibodies raised against rat as well as human "liver" lipase as shown by inhibition experiments. These results show that Hep G2 cells secrete "liver" lipase and that there seems to exist a structural homology between the lipases from rat and human origin.     

Studies on lipogenesis in vivo: Fatty acid and cholesterol synthesis in hyperglycaemic-obese mice

1. Lipogenesis has been studied in intact genetically obese mice by measuring the incorporation of a single oral dose of 250mg. of [U-¹⁴C]glucose into fatty acid and cholesterol in the liver and extrahepatic tissues. Studies were also carried out with [U-¹⁴C]glucose added to the diet and fed for 24hr. With either method of isotope administration, the conversion of [U-¹⁴C]glucose into fatty acid was greatly elevated in the livers of the obese mice. In contrast, conversion of the single dose of [¹⁴C]glucose into fatty acid in extrahepatic tissues of obese mice was only half that occurring in the non-obese litter mates. When [¹⁴C]glucose was given in the diet for 24hr. the total accumulation of labelled fatty acid in extrahepatic tissues of obese mice was slightly less than in the non-obese. Uptake of labelled glucose and conversion into fatty acid in adipose tissue of the obese mice decreased with age. 2. Conversion of the single dose of [¹⁴C]glucose into liver cholesterol was comparable in obese and non-obese mice fed on a purified low-fat diet. However, obese mice given this diet for 12 weeks accumulated 1·54% of cholesterol in the liver compared with 0·29% in the non-obese litter mates. This accumulation apparently resulted from a decrease in removal of cholesterol from the liver, rather than an increased synthesis. 3. Conversion of the single dose of [¹⁴C]glucose into extrahepatic fatty acid was decreased by 18hr. starvation proportionally as much in obese as in non-obese mice. The decrease in liver fatty acid synthesis caused by starvation also was considerable in obese mice, although somewhat less marked than in the non-obese. 4. The metabolic derangements in the liver could be more fundamental to the development of the obesity than the changes seen in extrahepatic tissues.