Role of kinesins in directed adenovirus transport and cytoplasmic exploration

Many viruses, including adenovirus, exhibit bidirectional transport along microtubules following cell entry. Cytoplasmic dynein is responsible for microtubule minus end transport of adenovirus capsids after endosomal escape. However, the identity and roles of the opposing plus end-directed motor(s) remain unknown. We performed an RNAi screen of 38 kinesins, which implicated Kif5B (kinesin-1 family) and additional minor kinesins in adenovirus 5 (Ad5) capsid translocation. Kif5B RNAi markedly increased centrosome accumulation of incoming Ad5 capsids in human A549 pulmonary epithelial cells within the first 30 min post infection, an effect dramatically enhanced by blocking Ad5 nuclear pore targeting using leptomycin B. The Kif5B RNAi phenotype was rescued by expression of RNAi-resistant Kif5A, B, or C, and Kif4A. Kif5B RNAi also inhibited a novel form of microtubule-based “assisted-diffusion” behavior which was apparent between 30 and 60 min p.i. We found the major capsid protein penton base (PB) to recruit kinesin-1, distinct from the hexon role we previously identified for cytoplasmic dynein binding. We propose that adenovirus uses independently recruited kinesin and dynein for directed transport and for a more random microtubule-based assisted diffusion behavior to fully explore the cytoplasm before docking at the nucleus, a mechanism of potential importance for physiological cargoes as well

Purification and proteomic identification of putative upstream regulators of polo-like kinase-1 from mitotic cell extracts

AbstractPolo-like kinase-1 (Plk1) is phosphorylated on Thr210 for activation during mitosis. Here, we investigated the question of which kinase(s) is the specific upstream kinase of mitotic Plk1. Upstream kinases of Plk1 were purified from mitotic cell extracts through column chromatography procedures, and identified by mass spectrometry. Candidates for Plk1 kinase included p21-activated kinase, aurora A, and mammalian Ste20-like kinases. Immunoprecipitates of these proteins from mitotic cell extracts phosphorylated Plk1 on Thr210. Even if the activity of Aurora A was blocked with a specific inhibitor, Plk1 phosphorylation still occurred, suggesting that function of Plk1 could be controlled by these kinases for proper mitotic progression, as well as by Aurora A in very late G2 phase for the beginning of mitosis.Structured abstractMINT-7996332: PAK1(uniprotkb:Q13153)physically interacts(MI:0915) withPLK1(uniprotkb:P53350) bypull down(MI:0096)MINT-7996345: PAK3(uniprotkb:O75914)physically interacts(MI:0915) withPLK1(uniprotkb:P53350) bypull down(MI:0096

A Case of Streptococcus gallolyticus subsp gallolyticus Infective Endocarditis with Colon Cancer: Identification by 16S Ribosomal DNA Sequencing

Although the association between Streptococcus bovis endocarditis and colon carcinoma is well known, very few cases of S. bovis infection associated with underlying malignancies have been reported in Korea The S. bovis group has been recently reclassified and renamed as Streptococcus gallolyticus and Streptococcus infantarius subspecies under a new nomenclature system. We report a case of infective endocarditis with colon cancer caused by S. gallolyticus subsp. gallolyticus (previously named S. bovis biotype 1). A 59-yr-old woman presented with a 1-month history of fever. Initial blood cultures were positive for gram-positive cocci, and echocardiography showed vegetation on mitral and aortic valves. Antibiotic treatment for infective endocarditis was started. The infecting strain was a catalase-negative and bile-esculin-positive alpha-hemolytic Streptococcus susceptible to penicillin and vancomycin. The strain was identified as S. gallolyticus subsp. gallolyticus with the use of the Vitek 2 GPI and API 20 Strep systems (bioMerieux, USA). The 16S rDNA sequences of the blood culture isolates showed 100% homology with those of S. gallolyticus subsp. gallolyticus reported in GenBank. The identification of the infecting organism, and the subsequent communication among clinical microbiologists and physicians about the changed nomenclature, led to the detection of colon cancer. The patient recovered after treatment with antibiotics, valve surgery, and operation for colon cancer. This is the first report of biochemical and genetic identification of S. gallolyticus subsp. gallolyticus causing infective endocarditis associated with underlying colon cancer in a Korean patient. (Korean J Lib Med 2010;30:160-5)Herrero IA, 2002, J CLIN MICROBIOL, V40, P3848, DOI 10.1128/JCM.40.10.3848-3850.2002Beck M, 2008, J CLIN MICROBIOL, V46, P2966, DOI 10.1128/JCM.00078-08Poyart C, 2002, INT J SYST EVOL MICR, V52, P1247, DOI 10.1099/js.0.02044-0Clarridge JE, 2001, J CLIN MICROBIOL, V39, P1549KOH DW, 2001, KOREAN J GASTROINTES, V23, P503Schlegel L, 2000, INT J SYST EVOL MICR, V50, P1425Ellmerich S, 2000, CARCINOGENESIS, V21, P753KWACK KK, 2000, KOREAN J MED, V59, P198Devriese LA, 1998, J CLIN MICROBIOL, V36, P3520COYKENDALL AL, 1989, CLIN MICROBIOL REV, V2, P315Schlegel L, 2003, INT J SYST EVOL MICR, V53, P631, DOI 10.1099/ijs.0.02361-0UH Y, 2006, KOREAN J CLIN MICROB, V9, P36RUOFF KL, 1989, J CLIN MICROBIOL, V27, P305FARROW JAE, 1984, SYST APPL MICROBIOL, V5, P467WILSON WR, 1981, JAMA-J AM MED ASSOC, V245, P360KLEIN RS, 1979, ANN INTERN MED, V91, P560KLEIN RS, 1977, NEW ENGL J MED, V297, P800MC CW, 1951, J MED ASS STATE ALA, V21, P162

Super Helium-Rich Population and the Origin of Extreme Horizontal-Branch Stars in Globular Clusters

Recent observations for the color-magnitude diagrams (CMDs) of the massive globular cluster Omega Centauri have shown that it has a striking double main sequence (MS), with a minority population of bluer and fainter MS well separated from a majority population of MS stars. Here we confirm, with the most up-to-date Y2 isochrones, that this special feature can only be reproduced by assuming a large variation (Delta Y = 0.15) of primordial helium abundance among several distinct populations in this cluster. We further show that the same helium enhancement required for this special feature on the MS can by itself reproduce the extreme horizontal-branch (HB) stars observed in Omega Cen, which are hotter than normal HB stars. Similarly, the complex features on the HBs of other globular clusters, such as NGC 2808, are explained by large internal variations of helium abundance. Supporting evidence for the helium-rich population is also provided by the far-UV (FUV) observations of extreme HB stars in these clusters, where the enhancement of helium can naturally explain the observed fainter FUV luminosity for these stars. The presence of super helium-rich populations in some globular clusters suggests that the third parameter, other than metallicity and age, also influences CMD morphology of these clusters.Comment: 4 pages, 4 figures, accepted for publication in the Astrophysical Journal Letter

Ubiquitin C-terminal hydrolase-activity is involved in sperm acrosomal function and anti-polyspermy defense during porcine fertilization

The 26S proteasome, which is a multi-subunit protease with specificity for substrate proteins that are postranslationally modified by ubiquitination, has been implicated in acrosomal function and sperm-zona pellucida (ZP) penetration during mammalian fertilization. Ubiquitin C-terminal hydrolases (UCHs) are responsible for the removal of polyubiquitin chains during substrate priming for proteasomal proteolysis. The inhibition of deubiquitination increases the rate of proteasomal proteolysis. Consequently, we have hypothesized that inhibition of sperm acrosome-borne UCHs increases the rate of sperm-ZP penetration and polyspermy during porcine in vitro fertilization (IVF). Ubiquitin aldehyde (UA), which is a specific nonpermeating UCH inhibitor, significantly (P < 0.05) increased polyspermy during porcine IVF and reduced (P < 0.05) UCH enzymatic activity measured in motile boar spermatozoa using a specific fluorometric UCH substrate, ubiquitin-AMC. Antibodies against two closely related UCHs, UCHL1 and UCHL3, detected these UCHs in the oocyte cortex and on the sperm acrosome, respectively, and increased the rate of polyspermy during IVF, consistent with the UA-induced polyspermy surge. In the oocyte, UCHL3 was primarily associated with the meiotic spindle. Sperm-borne UCHL3 was localized to the acrosomal surface and coimmunoprecipitated with a peripheral acrosomal membrane protein, spermadhesin AQN1. Recombinant UCHs, UCHL3, and isopeptidase T reduced polyspermy when added to the fertilization medium. UCHL1 was detected in the oocyte cortex but not on the sperm surface, and was partially degraded 6-8 h after fertilization. Enucleated oocyte-somatic cell electrofusion caused polarized redistribution of cortical UCHL1. We conclude that sperm-acrosomal UCHs are involved in sperm-ZP interactions and antipolyspermy defense. Modulation of UCH activity could facilitate the management of polyspermy during IVF and provide insights into male infertility

SHM-Based Probabilistic Fatigue Life Prediction for Bridges Based on FE Model Updating

Fatigue life prediction for a bridge should be based on the current condition of the bridge, and various sources of uncertainty, such as material properties, anticipated vehicle loads and environmental conditions, make the prediction very challenging. This paper presents a new approach for probabilistic fatigue life prediction for bridges using finite element (FE) model updating based on structural health monitoring (SHM) data. Recently, various types of SHM systems have been used to monitor and evaluate the long-term structural performance of bridges. For example, SHM data can be used to estimate the degradation of an in-service bridge, which makes it possible to update the initial FE model. The proposed method consists of three steps: (1) identifying the modal properties of a bridge, such as mode shapes and natural frequencies, based on the ambient vibration under passing vehicles; (2) updating the structural parameters of an initial FE model using the identified modal properties; and (3) predicting the probabilistic fatigue life using the updated FE model. The proposed method is demonstrated by application to a numerical model of a bridge, and the impact of FE model updating on the bridge fatigue life is discussedclos

Arf6 controls retromer traffic and intracellular cholesterol distribution via a phosphoinositide-based mechanism

Small GTPases play a critical role in membrane traffic. Among them, Arf6 mediates transport to and from the plasma membrane, as well as phosphoinositide signalling and cholesterol homeostasis. Here we delineate the molecular basis for the link between Arf6 and cholesterol homeostasis using an inducible knockout (KO) model of mouse embryonic fibroblasts (MEFs). We find that accumulation of free cholesterol in the late endosomes/lysosomes of Arf6 KO MEFs results from mistrafficking of Niemann–Pick type C protein NPC2, a cargo of the cation-independent mannose-6-phosphate receptor (CI-M6PR). This is caused by a selective increase in an endosomal pool of phosphatidylinositol-4-phosphate (PI4P) and a perturbation of retromer, which controls the retrograde transport of CI-M6PR via sorting nexins, including the PI4P effector SNX6. Finally, reducing PI4P levels in KO MEFs through independent mechanisms rescues aberrant retromer tubulation and cholesterol mistrafficking. Our study highlights a phosphoinositide-based mechanism for control of cholesterol distribution via retromer

Sperm Proteasomes Degrade Sperm Receptor on the Egg Zona Pellucida during Mammalian Fertilization

Despite decades of research, the mechanism by which the fertilizing spermatozoon penetrates the mammalian vitelline membrane, the zona pellucida (ZP) remains one of the unexplained fundamental events of human/mammalian development. Evidence has been accumulating in support of the 26S proteasome as a candidate for echinoderm, ascidian and mammalian egg coat lysin. Monitoring ZP protein degradation by sperm during fertilization is nearly impossible because those few spermatozoa that penetrate the ZP leave behind a virtually untraceable residue of degraded proteins. We have overcome this hurdle by designing an experimentally consistent in vitro system in which live boar spermatozoa are co-incubated with ZP-proteins (ZPP) solubilized from porcine oocytes. Using this assay, mimicking sperm-egg interactions, we demonstrate that the sperm-borne proteasomes can degrade the sperm receptor protein ZPC. Upon coincubation with motile spermatozoa, the solubilized ZPP, which appear to be ubiquitinated, adhered to sperm acrosomal caps and induced acrosomal exocytosis/formation of the acrosomal shroud. The degradation of the sperm receptor protein ZPC was assessed by Western blotting band-densitometry and proteomics. A nearly identical pattern of sperm receptor degradation, evident already within the first 5 min of coincubation, was observed when the spermatozoa were replaced with the isolated, enzymatically active, sperm-derived proteasomes. ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis. Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates. Preincubation of intact zona-enclosed ova with isolated active sperm proteasomes caused digestion, abrasions and loosening of the exposed zonae, and significantly reduced the fertilization/polyspermy rates after IVF, accompanied by en-mass detachment of zona bound sperm. Thus, the sperm borne 26S proteasome is a candidate zona lysin in mammals. This new paradigm has implications for contraception and assisted reproductive technologies in humans, as well as animals