High-resolution imaging demonstrates dynein-based vesicular transport of activated Trk receptors

Target-derived neurotrophins signal from nerve endings to the cell body to influence cellular and nuclear responses. The retrograde signal is conveyed by neurotrophin receptors (Trks) themselves. To accomplish this, activated Trks may physically relocalize from nerve endings to the cell bodies. However, alternative signaling mechanisms may also be used. To identify the vehicle wherein the activated Trks are located and transported, and to identify associated motor proteins that would facilitate transport, we use activation-state specific antibodies in concert with immunoelectron microscopy and deconvolution microscopy. We show that the'activated Trks within rat sciatic nerve axons are preferentially localized to coated and uncoated vesicles. These vesicles are moving in a retrograde direction and so accumulate distal to a ligation site. The P-Trk containing vesicles, in turn, colocalize with dynein components, and not with kinesins. Collectively, these results indicate activated Trk within axons travel in vesicles and dynein is the motor that drives these vesicles towards the cell bodies.     

Lithium inhibits aluminum-induced apoptosis in rabbit hippocampus,by preventing cytochrome c translocation,Bcl-2 decrease,Bax elevation and caspase-3 activation

A variety of studies on neuronal death models suggest that lithium has neuroprotective properties. In the present investigation, we have examined the effect of chronic lithium treatment on hippocampus, as monitored by changes at the subcellular level of apoptosis-regulatory proteins which have been induced by the neurotoxin, aluminum maltolate. Intracisternal administration of aluminum into rabbit brain induces cytochrome c release, decreases levels of the anti-apoptotic proteins Bcl-2 and Bcl-X(L), increases levels of the pro-apoptotic Bax, activates caspase-3, and causes DNA fragmentation as measured by the TUNEL assay. Pretreatment for 14 days with 7 mm of lithium carbonate in drinking water prevents aluminum-induced translocation of cytochrome c, and up-regulates Bcl-2 and Bcl-X(L,) down-regulates Bax, abolishes caspase-3 activity and reduces DNA damage. The regulatory effect of lithium on the apoptosis-controlling proteins occurs in both the mitochondria and endoplasmic reticulum. We propose that the neuroprotective effect of lithium involves the modulation of apoptosis-regulatory proteins present in the subcellular organelles of rabbit brain.     

YIL113w encodes a functional dual-specificity protein phosphatase which specifically interacts with and inactivates the Slt2/Mpk1p MAP kinase in S. cerevisiae

We show here that the YIL113w gene of Saccharomyces cerevisiae encodes a functional protein phosphatase. Yil113p shows no activity in vitro towards either phosphorylated casein or myelin basic protein. However, Yil113p dephosphorylates activated extracellular signal-regulated kinase 2 MAP kinase indicating that it is a dual-specificity MAP kinase phosphatase. In support of this we find that Yil113p specifically interacts with the stress-activated Slt2/Mpk1p MAP kinase of S. cerevisiae. Furthermore, expression of Yil113p causes the dephosphorylation of Slt2/Mpk1p in vivo, while expression of an inactive mutant of Yil113p causes the accumulation of phosphorylated Slt2/Mpk1p. We conclude that the physiological target of YIL113p is Slt2/Mpk1p.     

Polytene chromosomes of Simulium craigi (Diptera: Simuliidae)

Simulium craigi Adler and Currie is a polymorphic species based on polytene chromosome banding patterns in the long arm of chromosome III (IIIL). Three cytotypes are described based on the predominant IIIL sequences. These correspond to three broad geographic areas: cytotype CC from Pennsylvania; cytotype AF from Ontario and Manitoba; and cytotype ACF/BCF from New Hampshire. In the absence of sympatric populations, these cytotype differences are best explained by clinal variation within a single species. The relationship of S. craigi to other described members of the S. vernum group is discussed.     

Protein kinase B is regulated in platelets by the collagen receptor glycoprotein VI

Phosphoinositide 3-kinase (PI3K) is a critical component of the signaling pathways that control the activation of platelets. Here we have examined the regulation of protein kinase B (PKB), a downstream effector of PI3K, by the platelet collagen receptor glycoprotein (GP) VI and thrombin receptors. Stimulation of platelets with collagen or convulxin (a selective GPVI agonist) resulted in PI3K-dependent, and aggregation independent, Ser(473) and Thr(308) phosphorylation of PKBalpha, which results in PKB activation. This was accompanied by translocation of PKB to cell membranes. The phosphoinositide-dependent kinase PDK1 is known to phosphorylate PKBalpha on Thr(308), although the identity of the kinase responsible for Ser(473) phosphorylation is less clear. One candidate that has been implicated as being responsible for Ser(473) phosphorylation, either directly or indirectly, is the integrin-linked kinase (ILK). In this study we have examined the interactions of PKB, PDK1, and ILK in resting and stimulated platelets. We demonstrate that in platelets PKB is physically associated with PDK1 and ILK. Furthermore, the association of PDK1 and ILK increases upon platelet stimulation. It would therefore appear that formation of a tertiary complex between PDK1, ILK, and PKB may be necessary for phosphorylation of PKB. These observations indicate that PKB participates in cell signaling downstream of the platelet collagen receptor GPVI. The role of PKB in collagen- and thrombin-stimulated platelets remains to be determined.     

Cross-reactivity of some antibodies to human epitopes with shrimp Pandalus borealis proteins: a possible aid in validation and characterization of crustacean cells in vitro

Cell characterization of primary cultures in vertebrates is well established but not in marine invertebrates. This fact is hampering advances in the development of tissue cultures from this species. In the present study, a panel of antibodies to structural proteins, stress proteins, oncogenes and proliferation antigens, developed against mammalian antigens, were tested in paraffin sections of the crustacean Pandalus borealis tissues. Several tissues were analysed: hepatopancreas, gills, ovaries, epithelium under the cuticle and abdominal muscle. Specific antibodies to crustacean proteins are not commercially available. The immunocytochemical results show that antibodies to human epitopes cross-react with antigens in the crustacean Pandalus borealis indicating that some cellular proteins are highly conserved in evolution. Cytokeratin, proliferating cell nuclear antigen, ras and p-glycoprotein were detected by immunocytochemistry in Pandalus borealis. No immunoreactivity for Ki-67 and metallothionein was observed. This system can help in validation and characterization of invertebrate cultures.     

Characterization of thermodynamic diversity between transmissible spongiform encephalopathy agent strains and its theoretical implications.

Some transmissible spongiform encephalopathy (TSE) (or "prion") strains, notably those derived from bovine spongiform encephalopathy, are highly resistant to total inactivation by heat. When three TSE strains derived from sheep with scrapie were heated, little inactivation took place at low temperatures, but at higher temperatures, considerable inactivation occurred. The temperature at which substantial inactivation first occurred varied according to TSE strain, and it was calculated to be 70 degrees C for the 22C strain, 84 degrees C for ME7, and 97 degrees C for 22A by fitting the data to a model based on competition between a destructive and a protective reaction. However, PrP(Sc) from mice infected with a range of TSE strains retained similar resistance to proteinase K digestion after heating to below or above these temperatures, showing that the properties of PrP(Sc) responsible for proteinase resistance do not correlate with those conferring thermostability on the TSE agent. The simplest explanation of these data is that the causal agent contains a macromolecular component that is structurally independent of the host, that it varies covalently between TSE strains, and that it is protected by other macromolecular components. The model is in accord with the virino hypothesis, which proposes a host-independent informational molecule protected by the host protein PrP.     

Construction and manipulation of an infectious clone of the bovine herpesvirus 1 genome maintained as a bacterial artificial chromosome

The complete genome of bovine herpesvirus 1 (BoHV-1) strain V155 has been cloned as a bacterial artificial chromosome (BAC). Following electroporation into Escherichia coli strain DH10B, the BoHV-1 BAC was stably propagated over multiple generations of its host. BAC DNA recovered from DH10B cells and transfected into bovine cells produced a cytopathic effect which was indistinguishable from that of the parent virus. Analysis of the replication kinetics of the viral progeny indicated that insertion of the BAC vector into the thymidine kinase gene did not affect viral replication. Specific manipulation of the BAC was demonstrated by deleting the gene encoding glycoprotein E by homologous recombination in DH10B cells facilitated by GET recombination. These studies illustrate that the propagation and manipulation of herpesviruses in bacterial systems will allow for rapid and accurate characterization of BoHV-1 genes. In turn, this will allow for the full utilization of BoHV-1 as a vaccine vector.