AIP1/ALIX is a binding partner for HIV-1 p6 and EIAV p9 functioning in virus budding

HIV-1 and other retroviruses exit infected cells by budding from the plasma membrane, a process requiring membrane fission. The primary late assembly (L) domain in the p6 region of HIV-1 Gag mediates the detachment of the virion by recruiting host Tsg101, a component of the class E vacuolar protein sorting (Vps) machinery. We now show that HIV Gag p6 contains a second region involved in L domain function that binds AIP1, a homolog of the yeast class E Vps protein Bro1. Further, AIP1 interacts with Tsg101 and homologs of a subunit of the yeast class E Vps protein complex ESCRT-III. AIP1 also binds to the L domain in EIAV p9, and this binding correlates perfectly with L domain function. These observations identify AIP1 as a component of the viral budding machinery, which serves to link a distinct region in the L domain of HIV-1 p6 and EIAV p9 to ESCRT-III.     

Gram Stains: A Resource for Retrospective Analysis of Bacterial Pathogens in Clinical Studies

We demonstrate the feasibility of using qPCR on DNA extracted from vaginal Gram stain slides to estimate the presence and relative abundance of specific bacterial pathogens. We first tested Gram stained slides spiked with a mix of 10⁸ cfu/ml of Escherichia coli and 10⁵ cfu/ml of Lactobacillus acidophilus. Primers were designed for amplification of total and species-specific bacterial DNA based on 16S ribosomal gene regions. Sample DNA was pre-amplified with nearly full length 16S rDNA ribosomal gene fragment, followed by quantitative PCR with genera and species-specific 16S rDNA primers. Pre-amplification PCR increased the bacterial amounts; relative proportions of Escherichia coli and Lactobacillus recovered from spiked slides remained unchanged. We applied this method to forty two archived Gram stained slides available from a clinical trial of cerclage in pregnant women at high risk of preterm birth. We found a high correlation between Nugent scores based on bacterial morphology of Lactobacillus, Gardenerella and Mobiluncus and amounts of quantitative PCR estimated genus specific DNA (rrn copies) from Gram stained slides. Testing of a convenience sample of eight paired vaginal swabs and Gram stains freshly collected from healthy women found similar qPCR generated estimates of Lactobacillus proportions from Gram stained slides and vaginal swabs. Archived Gram stained slides collected from large scale epidemiologic and clinical studies represent a valuable, untapped resource for research on the composition of bacterial communities that colonize human mucosal surfaces.     

A three-stage micropropagation system for a novel color morph of the wildflower, <Emphasis Type="Italic">Porteranthus trifoliatus</Emphasis> (L.) Britt

A three-stage micropropagation system was devised for Porteranthus trifoliatus ‘Pink Profusion’, a cultivar distinguished from the wild type by its pink flowers, purple stems and darker reddish leaves. Nodes of young shoots were used as explants, disinfested, and placed on several different Murashige and Skoog’s (MS) media formulations containing two levels of indole-3-butyric acid (IBA) and four levels of benzylaminopurine (BAP) in a factorial combination. An optimal number of commercially-usable shoots was achieved with BAP concentrations between 10 μM and 30 μM, while the addition of IBA made no significant difference in the number of shoots produced. Proliferated shoots could be rooted ex vitro without auxin treatment.     

Specific residues in the connector loop of the human cytomegalovirus DNA polymerase accessory protein UL44 are crucial for interaction with the UL54 catalytic subunit

The human cytomegalovirus DNA polymerase includes an accessory protein, UL44, which has been proposed to act as a processivity factor for the catalytic subunit, UL54. How UL44 interacts with UL54 has not yet been elucidated. The crystal structure of UL44 revealed the presence of a connector loop analogous to that of the processivity subunit of herpes simplex virus DNA polymerase, UL42, which is crucial for interaction with its cognate catalytic subunit, UL30. To investigate the role of the UL44 connector loop, we replaced each of its amino acids (amino acids 129 to 140) with alanine. We then tested the effect of each substitution on the UL44-UL54 interaction by glutathione S-transferase pulldown and isothermal titration calorimetry assays, on the stimulation of UL54-mediated long-chain DNA synthesis by UL44, and on the binding of UL44 to DNA-cellulose columns. Substitutions that affected residues 133 to 136 of the connector loop measurably impaired the UL44-UL54 interaction without altering the ability of UL44 to bind DNA. One substitution, I135A, completely disrupted the binding of UL44 to UL54 and inhibited the ability of UL44 to stimulate long-chain DNA synthesis by UL54. Thus, similar to the herpes simplex virus UL30-UL42 interaction, a residue of the connector loop of the accessory subunit is crucial for UL54-UL44 interaction. However, while alteration of a polar residue of the UL42 connector loop only partially reduced binding to UL30, substitution of a hydrophobic residue of UL44 completely disrupted the UL54-UL44 interaction. This information may aid the discovery of small-molecule inhibitors of the UL44-UL54 interaction.     

Unique substrate specificity of anaplastic lymphoma kinase (ALK): development of phosphoacceptor peptides for the assay of ALK activity

The anaplastic lymphoma kinase (ALK), whose constitutively active fusion proteins are responsible for 5-10% of non-Hodgkin's lymphomas, shares with the other members of the insulin receptor kinase (IRK) subfamily an activation loop (A-loop) with the triple tyrosine motif Y-x-x-x-Y-Y. However, the amino acid sequence of the ALK A-loop differs significantly from the sequences of both the IRK A-loop and the consensus A-loop for this kinase subfamily. A major difference is the presence of a unique "RAS" triplet between the first and second tyrosines of the ALK A-loop, which in IRK is replaced by "ETD". Here we show that a peptide reproducing the A-loop of ALK is readily phosphorylated by ALK, while a homologous IRK A-loop peptide is not unless its "ETD" triplet is substituted by "RAS". Phosphorylation occurs almost exclusively at the first tyrosine of the Y-x-x-x-Y-Y motif, as judged by Edman analysis of the phosphoradiolabeled product. Consequently, a peptide in which the first tyrosine had been replaced by phenylalanine (FYY) was almost unaffected by ALK. In contrast, a peptide in which the second and third tyrosines had been replaced by phenylalanine (YFF) was phosphorylated more rapidly than the parent peptide (YYY). A number of substitutions in the YFF peptide outlined the importance of Ile and Arg at positions n - 1 and n + 6 in addition to the central triplet, to ensure efficient phosphorylation by ALK. Such a peculiar substrate specificity allows the specific monitoring of ALK activity in crude extracts of NPM-ALK positive cells, using the YFF peptide, which is only marginally phosphorylated by a number of other tyrosine kinases.     

The membrane environment of endogenous cellular prion protein in primary rat cerebellar neurons

We studied the membrane environment of cellular prion protein in primary cultured rat cerebellar neurons differentiated in vitro. In these cells, about 45% of total cellular prion protein (corresponding to a 35-fold enrichment) is associated with a low-density, sphingolipid- and cholesterol-enriched membrane fraction, that can be separated by flotation on sucrose gradient. Biotinylation experiments indicated that almost all prion protein recovered in this fraction was exposed at the cell surface. Prion protein was efficiently separated from this fraction by a monoclonal antibody immuno-separation procedure. Under conditions designed to preserve lipid-mediated membrane organization, several proteins were found in the prion protein-enriched membrane domains (i.e. the non-receptor tyrosine kinases Lyn and Fyn and the neuronal glycosylphosphatidylinositol-anchored protein Thy-1). The prion protein-rich membrane domains contained, as well, about 50% of the sphingolipids, cholesterol and phosphatidylcholine present in the sphingolipid-enriched membrane fraction. All main sphingolipids, including sphingomyelin, neutral glycosphingolipids and gangliosides, were similarly enriched in the prion protein-rich membrane domains. Thus, prion protein plasma membrane environment in differentiated neurons resulted to be a complex entity, whose integrity requires a network of lipid-mediated non-covalent interactions.     

Expression, purification, and inhibition of human RET tyrosine kinase

Tyrosine kinases are emerging as frequent targets of primary oncogenic events and therefore represent an optimal focus of therapeutical intervention. Genetic alterations that cause dysregulated activation of the RET tyrosine kinase are responsible for a significant fraction of thyroid carcinomas. In an effort towards therapeutic RET inactivation, we have developed a method for expression and purification of recombinant RET catalytic domain for structural purposes and for use in the screening of potential inhibitors of RET kinase activity. His-tagged RET kinase domain was purified from Sf9 insect cell lysate using a two-step chromatographic protocol and characterised. Purified recombinant RET phosphorylated itself and exogenous substrates at physiological pH. A specific peptide substrate, derived from RET activation loop, was identified and experimentally validated. These reagents were used to develop a rapid ELISA-based kinase assay for screening potential inhibitors. Novel RET inhibitors were identified using this assay.     

Glycogen- and PP1c-targeting subunit GM is phosphorylated at Ser48 by sarcoplasmic reticulum-bound Ca2+-calmodulin protein kinase in rabbit fast twitch skeletal muscle

Multifunctional Ca(2+)-calmodulin-dependent protein kinase (CaMKII) is a Ser/Thr protein kinase uniformly distributed within the sarcoplasmic reticulum (SR) of skeletal muscle. In fast twitch muscle, no specific substrates of CaMKII have yet been identified in nonjunctional SR. Previous electron microscopy data showed that glycogen particles containing glycogen synthase (GS) associate with SR at the I band level. Furthermore, recent evidence implicates CaMKII in regulation of glucose and glycogen metabolism. Here, we demonstrate that the glycogen- and protein phosphatase 1-targeting subunit, also known as G(M), selectively localizes to the SR membranes of rabbit skeletal muscle and that G(M) and GS co-localize at the level of the I band. We further show that G(M), GS, and PP1c assemble in a structural complex that selectively localizes to nonjunctional SR and that G(M) is phosphorylated by SR-bound CaMKII and dephosphorylated by PP1c. On the other hand, no evidence for a structural interaction between G(M) and CaMKII was obtained. Using His-tagged G(M) recombinant fragments and site-directed mutagenesis, we demonstrate that the target of CaMKII is Ser(48). Taken together, these data suggest that SR-bound CaMKII participates in the regulation of GS activity through changes in the phosphorylation state of G(M). Based on these findings, we propose that SR-bound CaMKII participates in the regulation of glycogen metabolism, under physiological conditions involving repetitive raises elevations of [Ca(2+)](i).     

miR-15a and miR-16-1 down-regulation in pituitary adenomas

Micro RNAs (miRs) are small noncoding RNAs, functioning as antisense regulators of other RNAs. miR-15a and miR-16-1 genes are located at chromosome 13q14, a region which is frequently deleted in pituitary tumors. An inverse correlation has been shown in B cell chronic lymphocytic leukemia (B-CLL) between miR-15a and miR-16-1 expression and the expression levels of arginyl-tRNA synthetase (RARS), an enzyme which associates with the cofactor p43 in the aminoacyl-tRNA synthetase complex. When secreted, p43 regulates local inflammatory response and macrophage chemotaxis, and seems to have anti-neoplastic properties in mice. We explored miR-15a and miR-16-1 expression in 10 GH-secreting and in 10 PRL-secreting pituitary macroadenomas by Northern blot, and investigated the possible correlation with in vivo and in vitro characteristics. We found that miR-15a and miR-16-1 are expressed at lower levels in pituitary adenomas as compared to normal pituitary tissue. Moreover, their expression inversely correlates with tumor diameter and with RARS expression (P < 0.05), but directly correlates with p43 secretion (P < 0.02). Therefore, miR15 and miR16 down-regulation in pituitary adenomas correlates with a greater tumor diameter and a lower p43 secretion, suggesting that these genes may, at least in part, influence tumor growth.     

Global proteome discovery using an online three-dimensional LC-MS/MS

We have developed a proteomics technology featuring on-line three-dimensional liquid chromatography coupled to tandem mass spectrometry (3D LC-MS/MS). Using 3D LC-MS/MS, the yeast-soluble, urea-solubilized peripheral membrane and SDS-solubilized membrane protein samples collectively yielded 3019 unique yeast protein identifications with an average of 5.5 peptides per protein from the 6300-gene Saccharomyces Genome Database searched with SEQUEST. A single run of the urea-solubilized sample yielded 2255 unique protein identifications, suggesting high peak capacity and resolving power of 3D LC-MS/MS. After precipitation of SDS from the digested membrane protein sample, 3D LC-MS/MS allowed the analysis of membrane proteins. Among 1221 proteins containing two or more predicted transmembrane domains, 495 such proteins were identified. The improved yeast proteome data allowed the mapping of many metabolic pathways and functional categories. The 3D LC-MS/MS technology provides a suitable tool for global proteome discovery.