A role for the Saccharomyces cerevisiae RENT complex protein Net1 in HMR silencing

Silencing in the yeast Saccharomyces cerevisiae is known in three classes of loci: in the silent mating-type loci HML and HMR, in subtelomeric regions, and in the highly repetitive rDNA locus, which resides in the nucleolus. rDNA silencing differs markedly from the other two classes of silencing in that it requires a DNA-associated protein complex termed RENT. The Net1 protein, a central component of RENT, is required for nucleolar integrity and the control of exit from mitosis. Another RENT component is the NAD(+)-dependent histone deacetylase Sir2, which is the only silencing factor known to be shared among the three classes of silencing. Here, we investigated the role of Net1 in HMR silencing. The mutation net1-1, as well as NET1 expression from a 2micro-plasmid, restored repression at silencing-defective HMR loci. Both effects were strictly dependent on the Sir proteins. We found overexpressed Net1 protein to be directly associated with the HMR-E silencer, suggesting that Net1 could interact with silencer binding proteins and recruit other silencing factors to the silencer. In agreement with this, Net1 provided ORC-dependent, Sir1-independent silencing when artificially tethered to the silencer. In contrast, our data suggested that net1-1 acted indirectly in HMR silencing by releasing Sir2 from the nucleolus, thus shifting the internal competition for Sir2 from the silenced loci toward HMR.     

LC-NMR applied to the characterisation of cardiac glycosides from three micropropagated Isoplexis species

Species of the genus Isoplexis are of particular interest with respect to the biochemical pathway leading to the cardenolides. It is important to determine whether or not 5beta-configured compounds, typically produced by Digitalis species and used in medicine, are present together with their respective alpha-isomers in Isoplexis spp. Structure elucidation by LC-NMR of the products isolated from in vitro regenerated Isoplexis canariensis, I. chalcantha and I. isabelliana was carried out, and similarities were observed among the products of the three species, including the presence of digitoxigenin-type cardenolides in I. canariensis and xysmalogenin and canarigenin derivatives in I. chalcantha never previously reported in these species. Pregnane glycosides not found until now either in Isoplexis or Digitalis were also detected.     

Expression of peroxisomal proteins provides clear evidence for the presence of peroxisomes in the male germ cell line GC1spg

Peroxisomes are cell organelles that perform multiple functions in the metabolism of lipids and of reactive oxygen species. They are present in most eukaryotic cells. However, they are believed to be absent in spermatozoa and they have never been described in male germ cells. We have used the immortalized germ cell line GC1spg to investigate the expression of peroxisomal proteins in germ cells of mice. The GC1spg cells represent the differentiation state of type B spermatogonia or preleptotene spermatocytes. We could show that peroxisomal membrane proteins like Pmp70 and Pex14p as well as peroxisomal matrix proteins like catalase or acyl CoA oxidase are expressed in GC1spg cells. All these proteins were colocalized in the same structures within the cells. Furthermore, by electron microscopy we have identified subcellular particles with an ultrastructural appearance that is characteristic of peroxisomes. This is the first report demonstrating the peroxisomal compartment in male germ cells of mice.     

The DNA binding and pairing preferences of the archaeal RadA protein demonstrate a universal characteristic of DNA strand exchange proteins

The archaeal RadA protein is a homologue of the Escherichia coli RecA and Saccharomyces cerevisiae Rad51 proteins and possesses the same biochemical activities. Here, using in vitro selection, we show that the Sulfolobus solfataricus RadA protein displays the same preference as its homologues for binding to DNA sequences that are rich in G residues, and under-represented in A and C residues. The RadA protein also displays enhanced pairing activity with these in vitro-selected sequences. These parallels between the archaeal, eukaryal and bacterial proteins further extend the universal characteristics of DNA strand exchange proteins.     

Novel allocolchicinoids with an eight membered B-ring: design, synthesis and inhibition of tubulin assembly

Several B-ring variations of O-methyl androbiphenyline (8), newly accessible from (-)-(M,7S)-colchicine via photooxygenation and subsequent endoperoxide-transformation, were synthesized and evaluated for their inhibitory effects on tubulin assembly in vitro. The amino-allocolchicinoid (9), a key compound in this study, was transformed to the highly potent ketone 10 and by oxidation with H2O2/Na2WO4 to a mixture of syn/anti-oximes, like 11 and 12. These could easily be transformed to hitherto unknown allocolchicinoids 13 and 14 with an eight membered B-ring lactam obtained via a Beckmann rearrangement. Surprisingly both do not notably affect tubulin assembly, despite obvious structural similarities with active analogues of the thiocolchicine- and azasteganacin-series.     

Localization of fibroblast growth factor 2 (FGF-2) protein and the receptors FGFR 1–4 in normal human seminiferous epithelium

 Fibroblast growth factor 2 (FGF-2), which occurs in various isoforms both species and tissue specifically, regulates cell proliferation and differentiation via a dual receptor system consisting of heparan sulphate proteoglycans and receptor tyrosine kinases (FGFRs). This study demonstrates for the first time the distribution pattern of FGF-2 and the receptors FGFR 1–4 in the normal seminiferous epithelium of adult men. In western blot analyses, the polyclonal antibody, anti-FGF-2, shows two immunoreactive bands at 18 and 24 kDa. On paraffin sections, positive immunoreaction occurs within the cytoplasm of spermatogonia. The distribution pattern of the polyclonal anti-FGFR 1–4 antibodies is as follows: anti-FGFR-1 (one 68-kDa band) stains nuclei and cytoplasm of spermatogonia; anti-FGFR-3 (five bands at 68, 78, 105, 125 and 145 kDa) stains the nuclei of all germ cells except those of elongated spermatids; and anti-FGFR-4 (one 48-kDa band) stains the cytoplasm of primary pachytene spermatocytes. We were unable to demonstrate FGFR-2 immunoreactivity either in western blot analysis or on paraffin sections. This distribution pattern suggests that FGF-2 in spermatogonia is involved in the autocrine and paracrine regulation of the proliferation and differentiation of spermatogonia and spermatocytes via the receptors FGFR-1, FGFR-3 and FGFR-4. Accepted: 23 December 1997     

Methods for homocysteine analysis and biological relevance of the results

It is now widely accepted that increased total plasma homocysteine (tHcy) is a risk factor for cardiovascular disease. Hyperhomocysteinemia can be caused by impaired enzyme function as a result of genetic mutation or vitamin B (B(2), B(6), B(9), B(12)) deficiency. A lot of methods are now available for tHcy determination. High-pressure liquid chromatography (HPLC) with fluorescence detection are at present the most widely used methods but immunoassays, easier to use, begin to supplant in-house laboratory methods. In order to help with the choice of a main relevant homocysteine analytical method, the characteristics, performances and limits of the main current methods are reviewed. One major drawback among all these available methods is the transferability which is not clearly established to date. The impact of both inter-method and inter-laboratory variations on the interpretation of the tHcy results are discussed.     

Sound localization behavior of the green treefrog (Hyla cinerea) and the barking treefrog (H. gratiosa)

Summary Female treefrogs (Hyla cinerea andH. gratiosa) can accurately localize a sound source (playback of male mating calls) if both ears are intact. When the sensitivity of one eardrum is attenuated, by coating it with a thin layer of silicone grease, females no longer can locate the sound source. This study demonstrates that female anurans rely on interaural cues for localization of a calling male. The neural basis for an anuran's sound localization ability presumably involves binaural convergence on single cells in the central auditory nervous system.This work was supported by research grants from the National Science Foundation and the U.S. Public Health Service. The assistance of Anne J.M. Moffat in measuring the directional characteristics of the loudspeaker is gratefully appreciated.     

Consumer versus resource control and the importance of habitat heterogeneity for estuarine bivalves

The relative influence of consumers (top down) and resources (bottom up) on the distribution and abundance of organisms remains a key question in ecology. We examined the relationships between consumer and resource variables along a productivity gradient for a dominant predator–prey interaction in a marine soft‐sediment system. We 1) quantified density and size of the clam Macoma balthica (prey species) in six replicate sites at each of four habitat types (shallow mud, deep mud, muddy sand and detrital mud) in the Rhode River, Chesapeake Bay. We selected one habitat type of high food availability and clam density (shallow mud) and another of low food availability and clam density (muddy sand) for manipulative experiments. Then, we 2) measured M. balthica survival and growth through transplants, 3) measured food availability as sedimentary organic carbon content, 4) quantified predator density, and 5) calculated predator foraging efficiency in the two habitat types. Clam density in the four habitat types differed and was related to sedimentary carbon availability and predator density. One of the habitats, detrital mud, appeared to be a population sink because it only held juvenile Macoma that never survived to reproductive age. Macoma size and growth, and predator (mainly blue crab Callinectes sapidus) densities were positively correlated with productivity and were higher in shallow mud than muddy sand. In contrast, Macoma mortality, local ‘interaction strength’, and predator foraging efficiency were lower in the productive habitat (shallow mud). Thus, predation intensity was inversely correlated with productivity (food availability); consumer and resource effects differed by habitat type; and, at a relatively small spatial scale, consumer and resource forces jointly determined population dynamics in this soft‐sediment marine system.