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1.
We have evaluated codon usage bias in Drosophila histone genes and have
obtained the nucleotide sequence of a 5,161-bp D. hydei histone gene repeat
unit. This repeat contains genes for all five histone proteins (H1, H2a,
H2b, H3, and H4) and differs from the previously reported one by a second
EcoRI site. These D. hydei repeats have been aligned to each other and to
the 5.0-kb (i.e., long) and 4.8-kb (i.e., short) histone repeat types from
D. melanogaster. In each species, base composition at synonymous sites is
similar to the average genomic composition and approaches that in the small
intergenic spacers of the histone gene repeats. Accumulation of synonymous
changes at synonymous sites after the species diverged is quite high. Both
of these features are consistent with the relatively low codon usage bias
observed in these genes when compared with other Drosophila genes. Thus,
the generalization that abundantly expressed genes in Drosophila have high
codon bias and low rates of silent substitution does not hold for the
histone genes.
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2.
Responding to the concern from our faculty that undergraduate students do not have robust laboratory skills, we designed and implemented a strategy to individually teach and assess the manipulative skills of students in first-year laboratories. Five core laboratory skills were selected for the course entitled Human Biology, a large, first-year class of students, most of whom were enrolled in Bachelor of Pharmacy and Human Movement Studies. Here, we report details for the 365 students enrolled primarily in Pharmacy and Human Movement Studies bachelor degree programs in semester 1 of 2006. We designed a specific strategy to assess five core laboratory skills: 1) accurate and precise use of a micropipette, 2) calculation of dilutions and preparation of diluted samples of saline, 3) accurate representation of data using a graph, 4) use of a light microscope, and 5) acquisition of digital data by measuring the latent period for the Achilles reflex. Graduate tutors were trained to teach and assess each student on each skill. The development of competency was tracked for all students across all five skills. Most students demonstrated proficiency on their first attempt. The development of proficiency across the core skills depended on both the skill and degree program. In semester 2 of 2006, 854 students mostly enrolled in the Bachelor of Science degree program and were similarly taught and assessed on the same five core skills. This approach was an effective teaching and assessment strategy that, when applied beyond first year, should increase the level of laboratory skills across undergraduate programs in physiology. 相似文献
3.
Nedd4-2 functionally interacts with ClC-5: involvement in constitutive albumin endocytosis in proximal tubule cells 总被引:4,自引:0,他引:4
Hryciw DH Ekberg J Lee A Lensink IL Kumar S Guggino WB Cook DI Pollock CA Poronnik P 《The Journal of biological chemistry》2004,279(53):54996-55007
Constitutive albumin uptake by the proximal tubule is achieved by a receptor-mediated process in which the Cl(-) channel, ClC-5, plays an obligate role. Here we investigated the functional interaction between ClC-5 and ubiquitin ligases Nedd4 and Nedd4-2 and their role in albumin uptake in opossum kidney proximal tubule (OK) cells. In vivo immunoprecipitation using an anti-HECT antibody demonstrated that ClC-5 bound to ubiquitin ligases, whereas glutathione S-transferase pull-downs confirmed that the C terminus of ClC-5 bound both Nedd4 and Nedd4-2. Nedd4-2 alone was able to alter ClC-5 currents in Xenopus oocytes by decreasing cell surface expression of ClC-5. In OK cells, a physiological concentration of albumin (10 mug/ml) rapidly increased cell surface expression of ClC-5, which was also accompanied by the ubiquitination of ClC-5. Albumin uptake was reduced by inhibiting either the lysosome or proteasome. Total levels of Nedd4-2 and proteasome activity also increased rapidly in response to albumin. Overexpression of ligase defective Nedd4-2 or knockdown of endogenous Nedd4-2 with small interfering RNA resulted in significant decreases in albumin uptake. In contrast, pathophysiological concentrations of albumin (100 and 1000 mug/ml) reduced the levels of ClC-5 and Nedd4-2 and the activity of the proteasome to the levels seen in the absence of albumin. These data demonstrate that normal constitutive uptake of albumin by the proximal tubule requires Nedd4-2, which may act via ubiquitination to shunt ClC-5 into the endocytic pathway. 相似文献
4.
5.
MacDonald JI Dietrich A Gamble S Hryciw T Grant RI Meakin SO 《Journal of neurochemistry》2012,121(6):861-880
Vesicular transport in neurons plays a vital role in neuronal function and survival. Nesca is a novel protein that we previously identified and herein describe its pattern of expression, subcellular localization and protein-protein interactions both in vitro and in vivo. Specifically, a large proportion of Nesca is in tight association with both actin and microtubule cytoskeletal proteins. Nesca binds to F-actin, microtubules, βIII and acetylated α-tubulin, but not neurofilaments or the actin-binding protein drebrin, in in vitro-binding assays. Nesca co-immunoprecipitates with kinesin heavy chain (KIF5B) and kinesin light-chain motors as well as with the synaptic membrane precursor protein, syntaxin-1, and is a constituent of the post-synaptic density. Moreover, in vitro-binding assays indicate that Nesca directly binds KIF5B, kinesin light-chain and syntaxin-1. In contrast, Nesca does not co-immunoprecipitate with the kinesin motors KIF1B, KIF3A nor does it bind syntaxin-4 or the synaptosome-associated protein 25 kDa (SNAP-25) in vitro. Nesca expression in neurons is highly punctuate, co-stains with syntaxin-1, and is found in fractions containing markers of early endosomes and Golgi suggesting that it is involved in vesicular transport. Collectively, these data suggest that Nesca functions as an adapter involved in neuronal vesicular transport including vesicles containing soluble N-ethylmaleimide sensitive factor attachment protein receptors that are essential to exocytosis. 相似文献
6.
Fucose is a major constituent of the protein- and lipid-linked glycans of
the various life-cycle stages of schistosomes. These fucosylated glycans
are highly antigenic and seem to play a role in the pathology of
schistosomiasis. In this article we describe the identification and
characterization of two fucosyltransferases (FucTs) in cercariae of the
avian schistosome Trichobilharzia ocellata, a GDP-Fuc:[Galbeta1--
>4]GlcNAcbeta-R alpha1-->3-FucT and a novel GDP-Fuc:Fucalpha-R
alpha1-- >2-FucT. Triton X-100 extracts of cercariae were assayed for
FucT activity using a variety of acceptor substrates. Type 1 chain
(Galbeta1- ->3GlcNAc) based compounds were poor acceptors, whereas those
based on a type 2 chain (Galbeta1-->4GlcNAc), whether
alpha2'-fucosylated, alpha3'-sialylated, or unsubstituted, and whether
present as oligosaccharide or contained in a glycopeptide or glycoprotein,
all served as acceptor substrates. In this respect the schistosomal alpha3-
FucT resembles human FucT V and VI rather than other known FucTs. N-
ethylmaleimide, an inhibitor of several human FucTs, had no effect on the
activity of the schistosomal alpha3-FucT, whereas GDP-beta-S was strongly
inhibitory. Large scale incubations were carried out with
Galbeta1-->4GlcNAc, GalNAcbeta1-->4GlcNAcbeta-O -(CH2)8COOCH3 and
Fucalpha1-->3GlcNAcbeta1-->2Man as acceptor substrates and the
products of the incubations were isolated using a sequence of
chromatographic techniques. By methylation analysis and 2D-TOCSY and
ROESY1H-NMR spectroscopy the products formed were shown to be Galbeta1--
>4[Fucalpha1-->2Fucalpha1-->3]GlcNAc,
GalNAcbeta1-->4[Fucalpha1-- >2Fucalpha1-->3]GlcNAcbe
ta-O-(CH2)8COOCH3, and Fucalpha1-->2Fucalpha1--
>3GlcNAcbeta1-->2Man, respectively. It is concluded that the alpha2-
FucT and alpha3-FucT are involved in the biosynthesis of the (oligomeric)
Lewisx sequences and the Fucalpha1-->2Fucalpha1-->3GlcNAc structural
element that have been described on schistosomal glycoconjugates.
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7.
Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands 总被引:2,自引:2,他引:0
Britten CJ; van den Eijnden DH; McDowell W; Kelly VA; Witham SJ; Edbrooke MR; Bird MI; de Vries T; Smithers N 《Glycobiology》1998,8(4):321-327
The alpha3 fucosyltransferase, FucT-VII, is one of the key
glycosyltransferases involved in the biosynthesis of the sialyl Lewis X
(sLex) antigen on human leukocytes. The sialyl Lewis X antigen
(NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential
component of the recruitment of leukocytes to sites of inflammation,
mediating the primary interaction between circulating leukocytes and
activated endothelium. In order to characterize the enzymatic properties of
the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been
expressed in Trichoplusia ni insect cells. The enzyme is capable of
synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from
3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels
of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies
using fucosylated NeuAcalpha(2-3)-(Galbeta(1- 4)GlcNAc)3-Me acceptors
demonstrate that FucT-VII is able to synthesize both di-fucosylated and
tri-fucosylated structures from mono- fucosylated precursors, but
preferentially fucosylates the distal GlcNAc within a polylactosamine
chain. Furthermore, the rate of fucosylation of the internal GlcNAc
residues is reduced once fucose has been added to the distal GlcNAc. These
results indicate that FucT-VII is capable of generating complex selectin
ligands, in vitro , however the order of fucose addition to the lactosamine
chain affects the rate of selectin ligand synthesis.
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8.
Liu HY MacDonald JI Hryciw T Li C Meakin SO 《The Journal of biological chemistry》2005,280(20):19461-19471
The human tumorous imaginal disc 1 (TID1) proteins including TID1(L) and TID1(S), members of the DnaJ domain protein family, are involved in multiple intracellular signaling pathways such as apoptosis induction, cell proliferation, and survival. Here we report that TID1 associates with the Trk receptor tyrosine kinases and regulates nerve growth factor (NGF)-induced neurite outgrowth in PC12-derived nnr5 cells. Binding assays and transfection studies showed that the carboxyl-terminal end of TID1 (residues 224-429) bound to Trk at the activation loop (Tyr(P)(683)-Tyr(684)(P)(684) in rat TrkA) and that TID1 was tyrosine phosphorylated by Trk both in yeast and in transfected cells. Moreover endogenous TID1 was also tyrosine phosphorylated by and co-immunoprecipitated with Trk in neurotrophin-stimulated primary rat hippocampal neurons. Overexpression studies showed that both TID1(L) and TID1(S) significantly facilitated NGF-induced neurite outgrowth in TrkA-expressing nnr5 cells possibly through a mechanism involving increased activation of mitogen-activated protein kinase. Consistently knockdown of endogenous TID1, mediated with specific short hairpin RNA, significantly reduced NGF-induced neurite growth in nnr5-TrkA cells. These data provide the first evidence that TID1 is a novel intracellular adaptor that interacts with the Trk receptor tyrosine kinases in an activity-dependent manner to facilitate Trk-dependent intracellular signaling. 相似文献
9.