Characterization of a mammalian Golgi-localized protein complex, COG, that is required for normal Golgi morphology and function

Multiprotein complexes are key determinants of Golgi apparatus structure and its capacity for intracellular transport and glycoprotein modification. Three complexes that have previously been partially characterized include (a) the Golgi transport complex (GTC), identified in an in vitro membrane transport assay, (b) the ldlCp complex, identified in analyses of CHO cell mutants with defects in Golgi-associated glycosylation reactions, and (c) the mammalian Sec34 complex, identified by homology to yeast Sec34p, implicated in vesicular transport. We show that these three complexes are identical and rename them the conserved oligomeric Golgi (COG) complex. The COG complex comprises four previously characterized proteins (Cog1/ldlBp, Cog2/ldlCp, Cog3/Sec34, and Cog5/GTC-90), three homologues of yeast Sec34/35 complex subunits (Cog4, -6, and -8), and a previously unidentified Golgi-associated protein (Cog7). EM of ldlB and ldlC mutants established that COG is required for normal Golgi morphology. "Deep etch" EM of purified COG revealed an approximately 37-nm-long structure comprised of two similarly sized globular domains connected by smaller extensions. Consideration of biochemical and genetic data for mammalian COG and its yeast homologue suggests a model for the subunit distribution within this complex, which plays critical roles in Golgi structure and function.     

Mechanism of glutamate stimulation of CO production in cerebral microvessels

Dilation of piglet pial arterioles to glutamate involves carbon monoxide (CO) produced from heme by heme oxygenase-2 (HO-2). Piglet cerebral microvessels and endothelial and smooth muscle cells grown on microcarrier beads were used to address the hypothesis that glutamate increases endothelial CO production by increasing HO-2 catalytic activity. CO was measured by gas chromatography/mass spectrometry. Glutamate increased CO production from endogenous heme by cerebral microvessels, endothelial cells, and smooth muscle cells. Glutamate increased the conversion of exogenous heme to CO. Protein tyrosine kinase inhibition blocked glutamate stimulation of CO production. Inhibition of protein tyrosine phosphatases stimulated CO production. Conversely, neither phorbol myristate acetate nor H-7 changed glutamate stimulation of CO production. The mechanism of HO-2 stimulation by glutamate appears to be independent of cytosolic Ca, because stimulation of CO production by glutamate was the same in Careplete medium, Ca-free medium with ionomycin, and Careplete medium with ionomycin. Therefore, glutamate appears to increase HO-2 catalytic activity in cerebral microvessels via a tyrosine kinase mediated pathway.     

Functional equivalence of structurally distinct ribosomes in the malaria parasite, Plasmodium berghei

Unlike most eukaryotes, many apicomplexan parasites contain only a few unlinked copies of ribosomal RNA (rRNA) genes. Based on stage-specific expression of these genes and structural differences among the rRNA molecules it has been suggested that Plasmodium spp. produce functionally different ribosomes in different developmental stages. This hypothesis was investigated through comparison of the structure of the large subunit rRNA molecules of the rodent malaria parasite, Plasmodium berghei, and by disruption of both of the rRNA gene units that are transcribed exclusively during development of this parasite in the mosquito (S-type rRNA gene units). In contrast to the human parasite, Plasmodium falciparum, we did not find evidence of structural differences in core regions of the distinct large subunit rRNAs which are known to be associated with catalytic activity including the GTPase site that varies in P. falciparum. Knockout P. berghei parasites lacking either of the S-type gene units were able to complete development in both the vertebrate and mosquito hosts. These results formally exclude the hypothesis that two functionally different ribosome types distinct from the predominantly blood stage-expressed A-type ribosomes, are required for development of all Plasmodium species in the mosquito. The maintenance of two functionally equivalent rRNA genes might now be explained as a gene dosage phenomenon.     

Primary structure of the Plasmodium vivax crk2 gene and interference of the yeast cell cycle upon its conditional expression

The cdc2 gene product, a 34-kDa protein kinase, plays a universal role in the M phase of the eukaryotic cell cycle. To study the cell cycle regulation in malarial parasites, we have characterized a cdc2-related gene from the most widely distributed human malaria, Plasmodium vivax (Pvcrk2). The full-length Pvcrk2 revealed 90--99% homology with Crk2 proteins from other Plasmodium species and approximately 60% homology with p34(cdc2) proteins from higher eukaryotes. We used the temperature-sensitive Schizosaccharomyces pombe cdc2 mutant (cdc2-33(ts)) for gene complementation studies. Expression of the full-length 33-kDa PvCrk2 protein, a truncated 27-kDa version, and two chimeric proteins in which we exchanged the N- and C-terminal regions of PvCrk2 with their S. pombe counterparts at the restrictive temperature in the mutant cdc2-33(ts) did not complement the cell cycle defect. However, conditional expression of the Pvcrk2 genes or the chimera containing the C terminus from Spcdc2 in mutant cdc2-33(ts) cells produced cell-cycle-arrested phenotypes only in the induced state and at the permissive temperature. Our results thus provide the first compelling genetic evidence that the plasmodial Crk2 gene product(s) is capable of interfering with the well-conserved eukaryotic cell cycle machinery.     

Prostaglandin E(2) regulates wound closure in airway epithelium

Repair of the airway epithelium after injury is critical for the maintenance of barrier function and the limitation of airway hyperreactivity. Airway epithelial cells (AECs) metabolize arachidonic acid to biologically active eicosanoids via the enzyme cyclooxygenase (COX). We investigated whether stimulating or inhibiting COX metabolites would affect wound closure in monolayers of cultured AECs. Inhibiting COX with indomethacin resulted in a dose-dependent inhibition of wound closure in human and feline AECs. Specific inhibitors for both COX-1 and COX-2 isoforms impaired wound healing. Inhibitors of 5-lipoxygenase did not affect wound closure in these cells. The addition of prostaglandin E(2) (PGE(2)) eliminated the inhibition due to indomethacin treatment, and the exogenous application of PGE(2) stimulated wound closure in a dose-dependent manner. Inhibition of COX with indomethacin only at initial time points resulted in a sustained inhibition of wound closure, indicating that prostanoids are involved in early wound repair processes such as spreading and migration. These differences in wound closure may be important if arachidonic acid metabolism and eicosanoid concentrations are altered in disease states such as asthma.     

Revision of Strunian crinoids and blastoids from Germany

In 1930W.E. Schmidt described all known crinoids from the German Early Carboniferous, including the Etroeungt beds of Germany, which are now judged to be latest Devonian (Famennian) in age. On a global basis, Famennian camerate crinoids generally show a closer relationship to succeeding Early Carboniferous faunas than they do to older Frasnian or Middle Devonian crinoids, which also is the case for the Strunian fauna. Holdovers from older Devonian faunas include, among others,Adelocrinus, a descendant of olderArthroacantha, in England and Germany, andPetaloblastus, which is one of the youngest genera of the blastoid family Hyperoblastidae. Precursors of younger Early Carboniferous groups include platycrinoids, primitive actinocrinoids, dichocrinoids, and the blastoid genusDoryblastus, which is one of the oldest members of the family Orbitremitidae. All of these groups, which became important parts of the Early Carboniferous crinoid and blastoid radiation, give Famennian crinoid faunas much more of an Early Carboniferous than a Devonian aspect. Rhipidocrinus schmidti n. sp. is erected for specimens that originally were reported from the Etroeungt asRhodocrinus uniarticulatus. We judge that there are currently four valid species assigned toRhipidocrinus: R. crenatus, R. perloricatus, R. praecursor, and our new species,R. schmidti. Hydriocrinus ratingensis Schmidt is reassigned to ?Sostronocrinus. We note thatSchmidt (1906), notJaekel (1906) as has been reported previously, is the author ofRhipidocrinus perloricatus. Owing to the poor preservation of the Etroeungt material, we regard the namePlatycrinites wunstorfi Schmidt 1930 to be a nomen nudum.     

Increased site 1 affinity improves biopotency of porcine growth hormone. Evidence against diffusion dependent receptor dimerization

Based on phage display optimization studies with human growth hormone (GH), it is thought that the biopotency of GH cannot be increased. This is proposed to be a result of the affinity of the first receptor for hormone far exceeding that which is required to trap the hormone long enough to allow diffusion of the second receptor to form the ternary complex, which initiates signaling. We report here that despite similar site 1 kinetics to the hGH/hGH receptor interaction, the potency of porcine GH for its receptor can be increased up to 5-fold by substituting hGH residues involved in site 1 binding into pGH. Based on extensive mutations and BIAcore studies, we show that the higher potency and site 1 affinity of hGH for the pGHR is primarily a result of a decreased off-rate associated with residues in the extended loop between helices 1 and 2 that interact with the two key tryptophans Trp104 and Trp169 in the receptor binding hot spot. Our mutagenic analysis has also identified a second determinant (Lys165), which in addition to His169, restricts the ability of non-primate hormones to activate hGH receptor. The increased biopotency of GH that we observe can be explained by a model for GH receptor activation where subunit alignment is critical for effective signaling.     

Induction of neutralizing antibodies in reindeer (Rangifer tarandus) after administration of a killed West Nile virus vaccine

In 2002, West Nile virus (WNV) infection with clinical neurologic disease and encephalomyelitis was described in reindeer (Rangifer tarandus). The susceptibility of reindeer to WNV prompted questions concerning vaccination of reindeer to prevent WNV infection. Between January and April 2003, eleven 2-4-yr-old, castrated male reindeer, some of which had antibody titers suggestive of prior exposure to WNV, were vaccinated three times at 4-wk intervals with a commercially available vaccine approved for use in horses. No adverse reactions to vaccination were noted. All vaccinated reindeer developed high neutralizing antibody titers to WNV, as determined by the plaque reduction neutralization test. Reindeer without antibody titers from previous natural exposure to WNV required a primary vaccination and one or two booster vaccinations for development of neutralizing antibody to WNV. Protective efficacy of vaccination was not evaluated. Vaccination of reindeer for WNV may be warranted in certain circumstances combined with management practices to limit exposure to potential vectors.