Modulation of the crayfish escape reflex--physiology and neuroethology

We review here factors that control the excitability of thegiant neuron-mediated tail-flip escape behavior in crayfish,focusing especially on recent findings concerning serotonergicmodulation. Serotonin can either facilitate or inhibit escapedepending on concentration and pattern of application. Low concentrationsfacilitate while high ones inhibit; however, if high concentrationsarise gradually they facilitate instead of inhibiting. The effectsof serotonin can also be altered by social experience, withapplication regimens that cause facilitation in social isolatescoming to produce inhibition after an extended period of livingas a subordinate. Attempts to understand both the possible physiologicalbasis of some of these complexities and their possible functionare discussed. Neuroethological investigations indicate thatgiant neuron-mediated escape is inhibited during the initialfights that establish social relationships and is facilitatedin their immediate aftermath. Once the relationship of a pairis well-established, the presence of the dominant tends to suppressgiant neuron-mediated escape (but not tail-flip escape mediatedby non-giant circuitry) in the subordinate, but the presenceof the subordinate has relatively little effect on the dominant.These patterns of modulation can be seen as consistent withthe known variations in serotonin's effect as a function ofconcentration and social experience and may provide a biologicalreason for these variations.     

SHIP-deficient mice are severely osteoporotic due to increased numbers of hyper-resorptive osteoclasts

The hematopoietic-restricted protein Src homology 2-containing inositol-5-phosphatase (SHIP) blunts phosphatidylinositol-3-kinase-initiated signaling by dephosphorylating its major substrate, phosphatidylinositol-3,4,5-trisphosphate. As SHIP(-/-) mice contain increased numbers of osteoclast precursors, that is, macrophages, we examined bones from these animals and found that osteoclast number is increased two-fold. This increased number is due to the prolonged life span of these cells and to hypersensitivity of precursors to macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappa B ligand (RANKL). Similar to pagetic osteoclasts, SHIP(-/-) osteoclasts are enlarged, containing upwards of 100 nuclei, and exhibit enhanced resorptive activity. Moreover, as in Paget disease, serum levels of interleukin-6 are markedly increased in SHIP(-/-) mice. Consistent with accelerated resorptive activity, 3D trabecular volume fraction, trabecular thickness, number and connectivity density of SHIP(-/-) long bones are reduced, resulting in a 22% loss of bone-mineral density and a 49% decrease in fracture energy. Thus, SHIP negatively regulates osteoclast formation and function and the absence of this enzyme results in severe osteoporosis.     

Cage use and feeding height preferences of captive common marmosets (Callithrix j. jacchus) in two-tier cages

Determining appropriate feeding regimes has important welfare implications for captive primates. This study examined the preference of food bowl heights in 6 pairs of common marmosets (Callithrix jacchus) housed in a 2-tier cage system. Given that marmosets are arboreal and spend most of their time in the upper half of their cages, we predicted that the marmosets would prefer a food bowl positioned at the top of the cage over one positioned at the bottom. We further predicted that this would be more apparent for the marmosets housed in lower tier than upper tier cages. Given a choice regarding where to feed, marmosets did prefer the top bowl to the bottom bowl; however, when only 1 food bowl was presented, its position had no significant effect on the marmosets' feeding behavior. In addition, contrary to the prediction, there were few differences in the marmosets' feeding behavior in the upper and lower tier cages. Feeding the marmosets in a bowl at the bottom of their cage did not result in greater cage use. On the basis of this study, we recommend positioning captive marmosets' food bowls high in the cage.     

Transcription corepressor CtBP is an NAD(+)-regulated dehydrogenase

The Lcd1p/Mec1p complex is crucial for normal S phase progression and for signaling DNA damage. We show that Lcd1p/Ddc2p and Mec1p in cell extracts bind to DNA ends. Although Lcd1p binds DNA independently of Mec1p, recruitment of Mec1p to DNA requires Lcd1p. DNA binding by Lcd1p is also independent of Rad9p, Rad17p, and Rad24p. Recombinant Lcd1p binds DNA, and this is impaired by Lcd1p mutations that abrogate its in vivo functions. Furthermore, Mec1p is recruited to cdc13-induced DNA damage and HO endonuclease-induced double-strand breaks in vivo. This requires Lcd1p, and recruitment of Lcd1p/Mec1p to cdc13-induced damage is abolished by Lcd1p mutations that abrogate its in vivo functions. Recruitment of Lcd1p to these lesions is independent of Mec1p and Rad9p/Rad24p. Thus, recruitment of Mec1p to DNA lesions by Lcd1p is crucial for the DNA damage response.     

Purification of pimplin,a paralytic heterodimeric polypeptide from venom of the parasitoid wasp Pimpla hypochondriaca,and cloning of the cDNA encoding one of the subunits

We have previously detected a paralytic factor in gel filtration-separated venom from the endoparasitoid wasp Pimpla hypochondriaca which is active against the fly Musca domestica. Now we have further purified this factor, which we have called pimplin, by reverse phase chromatography, and established using SDS-PAGE that it has a molecular mass of approximately 22 kDa. A 40 ng dose of pimplin administered to adult M. domestica by intrahaemocoelic injection was sufficient to kill all flies tested. Treatment of pimplin with β-mercaptoethanol prior to SDS-PAGE analysis resulted in the appearance of two polypeptides of approximately 15 and 6 kDa, indicating that pimplin is a heterodimer whose polypeptides are linked through a disulphide bond. Subunit masses of 10.544 and 6.318 kDa were determined using MALDI-TOF analysis indicating that the larger subunit migrates anomalously in SDS-PAGE. Using an oligonucleotide probe designed from N-terminal sequence obtained for the 15 kDa polypeptide, we have isolated a cDNA (pim1) encoding this larger pimplin subunit. The N-terminal amino acid sequence of pim1 occurred 28 residues beyond a predicted signal peptide cleavage site, indicating that pim1 is synthesised as a pre-propolypeptide which is secreted and proteolytically cleaved to yield the mature polypeptide stored in the venom sac. Beginning at the fourth residue of the mature pim1 venom polypeptide is a stretch of 46 residues consisting of alternating prolines, the significance of which is discussed in terms of possible host processing.     

Antisense oligonucleotides delivered to the lysosome escape and actively inhibit the hepatitis B virus

The subcellular fate and activity in inhibiting the hepatitis B virus of free and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-phosphorothioate oligonucleotides were studied. Their internalization and subcellular fate were monitored with confocal microscopy. A fraction of the internalized free oligonucleotides escaped into the cytoplasm and nucleus of Hep G2 cells but were not active antiviral agents. Covalently attaching the oligonucleotides to the HPMA copolymers via nondegradable dipeptide GG spacers resulted in sequestering the oligonucleotides in vesicles after internalization. Conjugation of the oligonucleotides to an HPMA copolymer via a lysosomally cleavable tetrapeptide GFLG spacer resulted in release of the oligonucleotide in the lysosome and subsequent translocation into the cytoplasm and nucleus of the cells. The HPMA copolymer-oligonucleotide conjugate possessed antiviral activity, indicating that phosphorothioate oligonucleotides released from the carrier in the lysosome were able to escape into the cytoplasm and nucleus and remain active. The Hep G2 cells appeared to actively internalize the phosphorothioate oligonucleotides as oligonucleotide-HPMA copolymer conjugates were internalized to a greater extent than unconjugated polymers.     

Discrete forms of amylose are synthesized by isoforms of GBSSI in pea

Amyloses with distinct molecular masses are found in the starch of pea embryos compared with the starch of pea leaves. In pea embryos, a granule-bound starch synthase protein (GBSSIa) is required for the synthesis of a significant portion of the amylose. However, this protein seems to be insignificant in the synthesis of amylose in pea leaves. cDNA clones encoding a second isoform of GBSSI, GBSSIb, have been isolated from pea leaves. Comparison of GBSSIa and GBSSIb activities shows them to have distinct properties. These differences have been confirmed by the expression of GBSSIa and GBSSIb in the amylose-free mutant of potato. GBSSIa and GBSSIb make distinct forms of amylose that differ in their molecular mass. These differences in product specificity, coupled with differences in the tissues in which GBSSIa and GBSSIb are most active, explain the distinct forms of amylose found in different tissues of pea. The shorter form of amylose formed by GBSSIa confers less susceptibility to the retrogradation of starch pastes than the amylose formed by GBSSIb. The product specificity of GBSSIa could provide beneficial attributes to starches for food and nonfood uses.