SSCP-SNP in pearl millet—a new marker system for comparative genetics

A considerable array of genomic resources are in place in pearl millet, and marker-aided selection is already in use in the public breeding programme at ICRISAT. This paper describes experiments to extend these publicly available resources to a single nucleotide polymorphism (SNP)-based marker system. A new marker system, single-strand conformational polymorphism (SSCP)-SNP, was developed using annotated rice genomic sequences to initially predict the intron-exon borders in millet expressed sequence tags (ESTs) and then to design primers that would amplify across the introns. An adequate supply of millet ESTs was available for us to identify 299 homologues of single-copy rice genes in which the intron positions could be precisely predicted. PCR primers were then designed to amplify approximately 500-bp genomic fragments containing introns. Analysis of these fragments on SSCP gels revealed considerable polymorphism. A detailed DNA sequence analysis of variation at four of the SSCP-SNP loci over a panel of eight inbred genotypes showed complex patterns of variation, with about one SNP or indel (insertion-deletion) every 59 bp in the introns, but considerably fewer in the exons. About two-thirds of the variation was derived from SNPs and one-third from indels. Most haplotypes were detected by SSCP. As a marker system, SSCP-SNP has lower development costs than simple sequence repeats (SSRs), because much of the work is in silico, and similar deployment costs and through-put potential. The rates of polymorphism were lower but useable, with a mean PIC of 0.49 relative to 0.72 for SSRs in our eight inbred genotype panel screen. The major advantage of the system is in comparative applications. Syntenic information can be used to target SSCP-SNP markers to specific chromosomal regions or, conversely, SSCP-SNP markers can be used to unravel detailed syntenic relationships in specific parts of the genome. Finally, a preliminary analysis showed that the millet SSCP-SNP primers amplified in other cereals with a success rate of about 50%. There is also considerable potential to promote SSCP-SNP to a COS (conserved orthologous set) marker system for application across species by more specifically designing primers to precisely match the model genome sequence.Electronic Supplementary Material Supplementary material is available for this article at     

6-hydroxydopamine lesions in anuran amphibians: a new model system for Parkinson's disease?

We investigated the effects of dopamine depletion on acoustically guided behavior of anurans by conducting phonotaxis experiments with female gray treefrogs (Hyla versicolor) before and 90 min after bilateral injections of 3, 6, or 12 microg 6-hydroxydopamine (6-OHDA) into the telencephalic ventricles. In experiments with one loudspeaker playing back a standard artificial mating call, we analyzed the effects of 6-OHDA on phonotactic response time. In choice tests we measured the degree of distraction from the standard call (20 pulses/s) by three different variants with altered pulse-rate (30/s, 40/s, 60/s). Five days after experiments, brains were immunostained for tyrosine hydroxylase. Labeled neurons were counted in the suprachiasmatic nucleus, posterior tuberculum, interpeduncular nucleus, and locus coeruleus, and correlation between neuronal numbers and behavioral scores was tested. Response times increased together with 6-OHDA concentrations, which was mainly due to longer immobile periods before the animals started movement. In choice tests the most irrelevant stimulus (60/s) distracted 6-OHDA injected females from the standard stimulus, while sham injected controls were undistracted. The number of catecholaminergic neurons decreased with increasing 6-OHDA concentration in the suprachiasmatic nucleus, posterior tuberculum, and interpeduncular nucleus. The normalized number of immunoreactive neurons in the posterior tuberculum was positively correlated with phonotaxis scores in the one-speaker test, demonstrating that motor deficits are a function of tubercular cell loss. We conclude that bilateral 6-OHDA lesions in anuran amphibians cause motor (difficulty to start movements) as well as cognitive symptoms (higher distraction by irrelevant stimuli) that have also been described for human Parkinson patients.     

Ubiquitination of intracellular bacteria: a new bacteria-sensing system?

Ubiquitination is a protein modification generally used by cells to tag proteins that are destined for proteasomal degradation. In a recent article, Perrin et al. reported that the ubiquitination system has a role in the recognition of bacterial pathogens in the cytosol of mammalian cells. They showed that polyubiquitinated proteins accumulate on the surface of cytosolic Salmonella typhimurium. In macrophages, but not epithelial cells, proteasomes become associated with the surface of cytosolic bacteria. The authors proposed that the ubiquitin-proteasome machinery might be implicated indirectly in bacterial clearance.     

CEUSPEC — a computerized system for fetal home telemetry

We have developed a computerized system whereby the fetal heart rate can be recorded telemetrically from patients' homes, transmitted over conventional public telephone lines, and then computer-processed in real time in the obstetric unit.     

Polyoxometalate–biomolecule conjugates: A new approach to create hybrid drugs for cancer therapeutics

Some polyoxometalate (POM) clusters have demonstrated attractive anticancer properties. Unfortunately, their cytotoxicity upon normal cell is one of fateful side effects obstructing their further clinic application as inorganic drugs. In this communication, we report a new approach to create hybrid drugs potentially for cancer therapeutics. At first, the POM cluster bioconjugates were created by attaching the bioactive ligands on an amine grafted POM via simple amidation reaction. The cytotoxicity study with breast cancer cells (MCF-7 and MDA-MB-231) and non-cancerous breast epithelial cell (MCF-10A) showed that rationally selected ligands with cancer-cell targeting ability on POM–biomolecule conjugates can impart enhanced anti-tumor activity and selectivity, thus representing a new concept to develop novel POM–biomolecule hybrid drugs with the potential synergistic effect: increased bioactivity and lower side effect.     

Intracellular hyaluronan: a new frontier for inflammation?

A variety of obstacles have hindered the ultrastructural localization of hyaluronan (HA). These include a lack of adequate fixation techniques to prevent the loss of HA, the lack of highly sensitive and specific probes, and a lack of accessibility due to the masking of HA by HA-binding macromolecules such as proteoglycans and glycoproteins. Despite these problems, a number of studies, both biochemical and histochemical, have been published indicating that HA is not restricted to the extracellular milieu, but is also present intracellularly. This review focuses on the possible functions of intracellular HA, its potential relationships to extracellular HA structures, and implications for inflammatory processes.     

MicroTom—a high-throughput model transformation system for functional genomics

We have developed a high-throughput Agrobacterium-mediated transformation model system using both nptII and the 5-enolpyruvylshikimate-3-phosphate synthase gene from Agrobacterium tumefaciens strain CP4 (cp4) based selections in MicroTom, a miniature rapid-cycling cherry tomato variety. With the NPTII selection system, transformation frequency calculated as independent transgenic events per inoculated explant ranged from 24 to 80% with an average of 56%, in industrial production scale transformation experiments. For CP4, with glyphosate selection, the average transformation frequency was 57%. Stable transformation frequency was positively correlated with transient expression (R=0.85), and variable with the genes of interest. DNA integration and germline transformation were confirmed by biological assay, Southern Blot analysis, and R₁ phenotype segregation. Transgene expression was observed in leaf, root, stem, flower, and fruit tissues of the transgenic plants. Ninety-five percent of transgenic events coexpressed two introduced genes based on β-glucuronidase (GUS) and neonmycin phosphotransferase II (NPTII) expression. Seventy-five percent of transgenic events contained one to two copies of the introduced uidA (GUS) gene based on Southern analysis. Transgenic plants from the cotyledon explants to the transgenic plants transferred to soil were produced within about 2–3 months depending on the genes of interest. The utility of this MicroTom model transformation system for functional genomic studies, such as identification of genes related to important agricultural traits and gene function, is discussed.     

Pyrogallol red-vanadium complex—a new stain for electron microscopy

 We report on the application of a pyrogallol red-vanadium complex (PR-V) for ultracytochemical staining of proteinaceous structures in animal tissues and cell cultures. This dye may be used as a general purpose stain in electron microscopy. In contrast to osmium tetroxide, the price of the material is low and no toxic vapors are produced. The PR-V complex was prepared by addition of vanadium (IV) oxide sulfate to pyrogallol red dissolved in acetate buffer (pH 5.6). The formation of the complex was indicated by a color change from purple-red (λ_max=520 nm) to violet (λ_max=539 nm) which occurred at equimolar concentrations of the dye and the metal salt. Under these conditions PR-V was stable for several days. The mechanism of PR-V binding was checked in dot blots using different proteins as well as heparin for control. While heparin remained unstained, proteins were stained in a dose-dependent manner. Deamination of proteins with nitric oxide strongly reduced PR-V staining in dot blots as well as in cell cultures. Optimal staining results of animal cells and tissues were obtained in specimens that had been mildly fixed for at least 1 h or longer with a mixture of 0.1% glutaraldehyde and 1.0% paraformaldehyde dissolved in phosphate-buffered saline, pH 7.2, washed with acetate buffer, pH 5.6, and subsequently treated with PR-V in the presence of 50% ethanol at room temperature. Control specimens without PR-V but treated en bloc with uranyl acetate or sodium molybdate showed similar contrast but less details in the ultrastructure of the tissue. All specimens were embedded in epoxy resin and ultrathin sections were stained conventionally with uranyl and lead salt solutions. In electron micrographs, membrane-associated particles, stress fibers and filaments of the cell cortex, collagen fibrils, tight junctions and desmosomes, and other proteinaceous components were clearly visualized only in the PR-V-treated specimens. In conclusion, the ability to bind selectively and specifically to proteinaceous structures makes PR-V a versatile stain to study the localization and distribution of these structures in cells and tissues at the ultrastructural level. Accepted: 14 June 1996     

Yeast mitochondrial biogenesis: a model system for humans?

Recently, our knowledge of yeast mitochondrial biogenesis has considerably progressed. This concerns the import machinery that guides preproteins synthesized on the cytoplasmic ribosomes through the mitochondrial outer and inner membranes, as well as the inner membrane insertion machinery of mitochondrially encoded polypeptides, or the proteins participating in the assembly and quality control of the respiratory complexes and ATP synthase. More recently, two new fields have emerged, biosynthesis of the iron-sulfur clusters and dynamics of the mitochondrion. Many of the newly discovered yeast proteins have homologues in human mitochondria. Thus, Saccharomyces cerevisiae has proven a particularly suitable simple organism for approaching the molecular bases of a growing number of human mitochondrial diseases caused by mutations in nuclear genes identified by positional cloning.     

The natriuretic peptide system in eels: a key endocrine system for euryhalinity?

The natriuretic peptide system of a euryhaline teleost, the Japanese eel (Anguilla japonica), consists of three types of hormones [atrial natriuretic peptide (ANP), ventricular natriuretic peptide (VNP), and C-type natriuretic peptide (CNP)] and four types of receptors [natriuretic peptide receptors (NPR)-A, -B, -C, and -D]. Although ANP is recognized as a volume-regulating hormone that extrudes both Na(+) and water in mammals, ANP more specifically extrudes Na(+) in eels. Accumulating evidence shows that ANP is secreted in response to hypernatremia and acts to inhibit the uptake and to stimulate the excretion of Na(+) but not water, thereby promoting seawater (SW) adaptation. In fact, ANP is secreted immediately after transfer of eels to SW and ameliorates sudden increases in plasma Na(+) concentration through inhibition of drinking and intestinal absorption of NaCl. ANP also stimulates the secretion of cortisol, a long-acting hormone for SW adaptation, whereas ANP itself disappears quickly from the circulation. Thus ANP is a primary hormone responsible for the initial phase of SW adaptation. By contrast, CNP appears to be a hormone involved in freshwater (FW) adaptation. Recent data show that the gene expression of CNP and its specific receptor, NPR-B, is much enhanced in FW eels. In fact, CNP infusion increases (22)Na uptake from the environment in FW eels. These results show that ANP and CNP, despite high sequence identity, have opposite effects on salinity adaptation in eels. This difference apparently originates from the difference in their specific receptors, ANP for NPR-A and CNP for NPR-B. VNP may compensate the effects of ANP and CNP for adaptation to respective media, because it has high affinity to both receptors. On the basis of these data, the authors suggest that the natriuretic peptide system is a key endocrine system that allows this euryhaline fish to adapt to diverse osmotic environments, particularly in the initial phase of adaptation.     

VEGFR3: a new target for antiangiogenesis therapy?

VEGFR-3 signaling plays an important role in developmental, physiological, and pathological angiogenesis and lymphangiogenesis. Tammela et al. in Nature show that VEGFR-3, via Notch regulation, is present on endothelial tip cells and is critical to sprouting angiogenesis.