bluuebunny/crossref_metadata_2025 huggingface dataset:
Perceptual Grouping in Shape from Shading
Authors: Kawabe Takahiro , Miura Kayo
Published: June 2004

We report on the reversal of asymmetry in visual-search tasks with shaded items. Previous studies have suggested that the target of a bottom-lit disk among distractors of top-lit disks is detected in a rapid and parallel manner, but not vice versa. However, in this study, we have shown that the compound items of top-lit disks were searched more quickly than those composed of bottom-lit disks where the items had to be segregated from their background. By modulating the inter-element distances, we confirmed that the reversal of search asymmetry cannot be due to the grouping of items. Further, we showed that the regions of the top-lit disks were perceived as figure more consistently than those of bottom-lit disks. The results indicate that the boundary assignment to the compound items of the top-lit disks enhances the segregation of search items from the background, and that the search mechanism may access the relatively higher representation that includes figure – ground relations.
Pointing out of the Picture
Authors: Koenderink Jan J , van Doom Andrea J , Kappers Astrid M L , Todd James T
Published: June 2004

The eyes of portrayed people are often noticed to ‘follow you’ when you move with respect to a flat painting or photograph. We investigated this well-known effect through extensive measurements of pictorial relief and apparent orientation of the picture surface for a number of viewing conditions, including frontal and oblique views. We conclude that cases of both oblique and frontal viewing are very similar in that perception simply follows what is indicated by the proximal stimulus, even though this may imply that the (perceived) physical and pictorial spaces segregate. The effect of foreshortening then causes an apparent narrowing of pictorial objects. We find no evidence for any ‘correction’ mechanisms that might be specifically active in oblique viewing conditions.
Perceiving Distance: A Role of Effort and Intent
Authors: Witt Jessica K , Proffitt Dennis R , Epstein William
Published: June 2004

Perceiving egocentric distance is not only a function of the optical variables to which it relates, but also a function of people's current physiological potential to perform intended actions. In a set of experiments, we showed that, as the effort associated with walking increases, perceived distance increases if the perceiver intends to walk the extent, but not if the perceiver intends to throw. Conversely, as the effort associated with throwing increases, perceived distance increases if people intend to throw to the target, but not if they intend to walk. Perceiving distance combines the geometry of the world with our behavior goals and the potential of our body to achieve these goals.
Economic evaluation of beef cattle breeding schemes incorporating performance testing of young bulls for feed intake
Authors: Archer J. A. , Barwick S. A. , Graser H.-U.
Published: June 2004

A model beef cattle breeding scheme consisting of a breeding unit and a commercial unit was used to evaluate the impact on genetic gain and profitability of incorporating feed intake measurements as an additional selection criterion in breeding programmes. Costs incurred by the breeding unit were compared with returns generated in the commercial unit, with bulls from the breeding unit being used as sires in the commercial unit. Two different market objectives were considered — a grass-fed product for the Australian domestic market, and a grain-fed product for the Japanese market. Breeding units utilising either artificial insemination or natural service were also considered. A base scenario was modelled incorporating a range of criteria available to Australian cattle breeders. A second scenario incorporated selection of sires for the breeding unit using a 2-stage selection process, with a proportion of bulls selected after weaning for measurement of (residual) feed intake. Measurement of feed intake of bulls improved accuracy of breeding unit sire selection by 14–50% over the equivalent base scenario, and genetic gain in the breeding objective was improved for all scenarios, with gains ranging from 8 to 38% over the base scenario. After accounting for the cost of measuring feed intake ($150–450), additional profit was generated from inclusion of feed intake measurement on a proportion of bulls for all breeding schemes considered. Profit was generally maximised where 10–20% of bulls were selected at weaning for measurement of intake, with improvement in profit ranging from 9 to 33% when optimal numbers of bulls were selected for intake measurement.
Genetic variation in starch digestion in feedlot cattle and its association with residual feed intake
Authors: Channon A. F. , Rowe J. B. , Herd R. M.
Published: June 2004

Steer progeny of lines selected for either low residual feed intake (high efficiency) or high residual feed intake (low efficiency) were grown out in a feedlot and measurements of feed intake, liveweight gain and faecal parameters were combined to investigate associations with starch digestion. There were significant differences between sire progeny groups in faecal pH and faecal dry matter content indicating genetic differences in starch digestion. This variation occurred even after differences in dry matter intake were accounted for. Furthermore, there was a significant association between these faecal parameters and mid-parent Estimated Breeding Values for residual feed intake, rib (12/13th) fat depth and rump fat depth (Australian P8 site). This confirmed an association between parameters believed to describe starch digestion and genetic variation in important production traits. Low faecal pH and faecal dry matter content were associated with higher residual feed intake (i.e. lower efficiency).
Some mechanisms regulating nutrient utilisation in livestock during immune activation: an overview
Authors: Colditz I. G.
Published: June 2004

The pro-inflammatory cytokines, IL-1, IL-6, TNFα and IFN α/β, produced during immune activation and tissue injury, override control of nutrient utilisation by the hypothalamic-somatotropic axis. The many effects of these cytokines include induction of fever and sickness behaviour, reduced fatty acid uptake by adipose tissue, reduced protein synthesis and enhanced protein breakdown in skeletal muscle, and gluconeogenesis, increased fatty acid synthesis and synthesis of acute phase proteins in the liver. Resistance to the effects of insulin, GH and IGF-1 is induced in adipose tissues, liver and muscle, at least in part through induction by pro-inflammatory cytokines of SOCS proteins which inhibit signal transduction and activation of gene transcription via the JAK/STAT pathway. These homeorhetic changes mobilise nutrients to fuel host defence responses. While an understanding of the mechanisms contributing to the catabolic state have arisen largely from studies of sepsis, trauma and acute challenge with biological mediators of the acute phase response, recent evidence in livestock suggests that graded production of pro-inflammatory cytokines during challenge with pathogens or subclinical infection can induce an incremental reduction in nutrient accretion in products of commercial value from livestock. This relationship highlights the value of good hygiene and reduced stress to improved feed utilisation for growth.
Is it useful to define residual feed intake as a trait in animal breeding programs?
Authors: Werf J. H. J. van der
Published: June 2004

Residual feed intake is a linear function of feed intake, production and maintenance of liveweight, and as such is an attractive characteristic to use to represent production efficiency. The phenotypic and genetic parameters of residual feed intake can be written as a function of its constituent traits. Moreover, selection indices containing the constituent traits are equivalent with an index that includes residual feed intake. Therefore, definition of the term residual feed intake may be useful to interpret variation in production efficiency, but it does not help in obtaining a better selection response than selection on constituent traits alone. In fact, multiple trait genetic evaluation of constituent traits rather than residual feed intake is likely to be more accurate as this more appropriately accommodates different models for the constituent traits and missing data. For residual feed intake to reflect true biological efficiency in growing animals, it is important that feed intake and liveweight are accurately measured. Accounting for growth and body composition would significantly help in revealing between-animal variation in feed utilisation. Random regression models can be helpful in indicating variation in feed efficiency over the growth trajectory.
Effect of missing data on the estimate of average daily feed intake in beef cattle
Authors: Hebart M. L. , Pitchford W. S. , Arthur P. F. , Archer J. A. , Herd R. M. , Bottema C. D. K.
Published: June 2004

The effect of missing feed intake records on the estimate of feed intake in cattle over a 70-day test period was studied. Two datasets, containing several cattle breeds at different ages, were analysed. The first dataset comprised 300-day-old Angus cattle, while the second dataset consisted of 900-day-old Limousin and Jersey crossbred cattle. A first-, second- and third-order polynomial and a non-linear function were fitted to the intake data. The 3 polynomial functions showed minimal differences in their goodness of fit to the data, with the non-linear function explaining the least variation in intake. Both datasets were analysed over a 70-day test period and shortened tests were compared to the full 70-day test. Tests were shortened by randomly deleting intake records or periodically deleting data on a daily or weekly basis. The effect of missing data on feed intake estimates was evaluated using Pearsons correlation and t-tests. The results suggest that a greater proportion of data can be removed randomly and daily, compared with missing data in weekly blocks. However, when data are removed in weekly blocks, removal in the middle of the test period gives a more accurate estimate of feed intake. It appears that in young growing animals, estimation of daily feed intake is more sensitive to missing feed intake records than for older cattle.
The effect of preweaning growth restriction on the feed intake and efficiency of cattle on a grain-based diet before slaughter
Authors: Hennessy D. W. , Arthur P. F.
Published: June 2004

Records on 78 steers and heifers were used to determine relationships among different measures of growth, feed efficiency and carcass traits, and to evaluate the effects of 2 preweaning growth rates on growth and efficiency in a feedlot. The preweaning growth rates [g/day; 871 (high) or 466 (low)] were imposed on the steers and heifers for 115 days before they were weaned at a mean age of 220 days. They were 'backgrounded' for 7 months on high quality pastures before finishing on grain-based diets in a feedlot for 85 days. The cattle were then slaughtered and carcass measurements recorded. Estimates were made on 10 growth and feed efficiency indices during the feedlot, and 5 carcass traits were assessed at slaughter. Of the indices, the Kleiber ratio which does not require measurement of feed intake (r = 0.98), was highly correlated with relative growth rate (RGR), as was feed conversion ratio (FCR, r = –0.82), which includes feed intake in the calculation. Residual feed intake, calculated using the expected feed intake estimated from the dataset (RFIREG), was correlated (r = 0.62) with RFI calculated from Australian feeding standards (RFISCA), and with FCR (r = 0.45). FCR was not correlated with either carcass rib or hot P8 fat depths but was significantly correlated with carcass weight (0.39), dressing percentage (0.41) and eye muscle area (0.33). RFIREG was not correlated with any of the 5 carcass traits. Whilst the average daily gain in the feedlot was similar between the 2 preweaning groups (1.544 kg/day), the low group was significantly (P<0.05) lighter at feedlot entry (323 v. 376 kg). Daily feed intake was lower (P<0.01) in the low group (10.9 v. 11.8 kg) such that the FCR tended to be lower (P<0.10) relative to the high group. Differences between the preweaning treatment groups were not significant for the other measures of feed efficiency. The results indicate that early life nutritional restriction has some influence on FCR in the feedlot, but little effect on those feed efficiency measures, such as RFI, that account for energy required for both maintenance of liveweight and growth.
Feed intake and efficiency in beef cattle: overview of recent Australian research and challenges for the future
Authors: Arthur P. F. , Archer J. A. , Herd R. M.
Published: June 2004

In the last 10 years, there have been 3 major research and development projects in Australia on the efficiency of feed utilisation by beef cattle. The primary objective of these projects has been to examine individual animal variation in feed efficiency and its exploitation for genetic improvement in beef cattle. The results of these projects indicate that genetic variation in feed efficiency exists in Australian beef herds, that feed efficiency is moderately heritable and that the potential exists to reduce the cost of beef production through selection for efficient cattle. These results have been further developed for industry application through the generation of BREEDPLAN estimated breeding values for net (or residual) feed intake (a feed efficiency trait) for Angus and Hereford–Polled Hereford breeds. Although economic analyses have indicated substantial benefit from selection for feed efficiency, the high initial cost of identifying animals which are superior for feed efficiency is a barrier to rapid adoption of the technology. Developing cost-effective methods of implementing the feed efficiency technology is thus an on-going research activity. Challenges for the future include: the development and use of more sophisticated statistical analyses procedures (such as random regression) for feed intake and efficiency evaluation; development of accurate methods of assessing individual animal feed intake at pasture; the adoption of a whole-production system approach to feed utilisation; and better integration of the disciplines of genetics and nutrition. The outcomes from research in the efficiency of feed utilisation in beef cattle have wider applications, not only in other livestock species, but also in human energetics, such as the control of obesity.
Biological basis for variation in residual feed intake in beef cattle. 2. Synthesis of results following divergent selection
Authors: Richardson E. C. , Herd R. M.
Published: June 2004

Experiments on Angus steer progeny following a single generation of divergent selection for residual feed intake suggest that there are many physiological mechanisms contributing to variation in residual feed intake. Difference in energy retained in protein and fat accounted for only 5% of the difference in residual feed intake following divergent selection. Differences in digestion contributed (conservatively) 10% and feeding patterns 2% to the variation in residual feed intake. The heat increment of feeding contributed 9% and activity contributed 10%. Indirect measures of protein turnover suggest that protein turnover, tissue metabolism and stress contributed to at least 37% of the variation in residual feed intake. About 27% of the difference in residual feed intake was due to variation in other processes such as ion transport, not yet measured. It is hypothesised that susceptibility to stress is a key driver for many of the biological differences observed following divergent selection for residual feed intake in beef cattle. Further research is required to accurately quantify the effect of selection for improved residual feed intake on protein turnover, tissue metabolism and ion transport, and to confirm the association between stress susceptibility and residual feed intake in beef cattle.
Genetic improvement of lean meat growth and feed efficiency in pigs
Authors: Hermesch S.
Published: June 2004

Optimising selection procedures for feed efficiency and lean meat growth in pigs is important because feed costs form a high cost component of production. In this paper a number of breeding objectives used in pig industries are reviewed and the concept of the linear-plateau growth model is outlined. This concept allows optimisation of selection for feed intake of the growing pig taking the pigs potential for protein deposition into account. This is a first step towards further collaboration between geneticists and scientists from other disciplines in order to gain a better understanding of physiological consequences of selection. The necessity of recording individual feed intake in group housed pigs has led to the development of electronic feeders. These computerised systems provide additional data including repeated weight and feed intake records as well as feeding pattern traits. How to make best use of this extra information in breeding programmes needs to be explored further. Implications of testing pigs under ad libitum and restricted feeding are outlined and results from selection experiments and commercial group house environments are summarised. The comparison of testing procedures shows that restricted feeding benefits selection for lean meat growth. However, in commercial environments the benefits depend on how well variation in feed intake can be controlled. Testing procedures should be expanded to explain a larger part of variation in daily feed intake, as is currently the case. This unexplained variation may be related to the animal’s activity, social interactions between animals and response to various stressors that occur in commercial group housed environments.
Ferritin reactions: Direct identification of the site for the diferric peroxide reaction intermediate
Authors: Liu Xiaofeng , Theil Elizabeth C.
Published: May 2004

Ferritins managing iron–oxygen biochemistry in animals, plants, and microorganisms belong to the diiron carboxylate protein family and concentrate iron as ferric oxide ≈10
14
times above the ferric
K
s
. Ferritin iron (up to 4,500 atoms), used for iron cofactors and heme, or to trap DNA-damaging oxidants in microorganisms, is concentrated in the protein nanocage cavity (5–8 nm) formed during assembly of polypeptide subunits, 24 in maxiferritins and 12 in miniferritins/DNA protection during starvation proteins. Direct identification of ferritin ferroxidase (F
ox
) sites, complicated by multiple types of iron–ferritin interactions, is now achieved with chimeric proteins where putative F
ox
site residues were introduced singly and cumulatively into an inactive host, an L maxiferritin. A dimagnesium ferritin cocrystal model guided site design and the diferric peroxo F
ox
intermediates (
A
at 650 nm) monitored activity. Diferric peroxo formation in chimeric and WT proteins had similar
K
app
values and Hill coefficients. Catalytic activity required cooperative ferrous substrate binding to two sites A (E, EXXH) and B (E, QXXD). The weaker B sites in ferritin contrast with stronger B sites (E, EXXH) in diiron carboxylate oxygenases, explaining diferric oxo/hydroxo product release in ferritin vs. diiron cofactor retention in oxygenases. Codons for Q/H and D/E differ by single nucleotides, suggesting simple DNA mutations relate site B diiron substrate sites and diiron cofactor sites in proteins. The smaller
k
cat
values in chimeras indicate the absence of second-shell residues important for ferritin substrate–product channeling that, when identified, will outline the entire iron path from ferritin pores through the F
ox
site to the mineral cavity.
Total synthesis and structure of the ramoplanin A1 and A3 aglycons: Two minor components of the ramoplanin complex
Authors: Shin Dongwoo , Rew Yosup , Boger Dale L.
Published: June 2004

Ramoplanin is a potent antibiotic, first disclosed in 1984, that acts by inhibiting bacterial cell-wall biosynthesis. The original ramoplanin complex was shown to consist of a mixture of three closely related compounds, ramoplanin A1–A3, of which ramoplanin A2 is the most abundant. The structure of ramoplanin A2 was unambiguously established first through a series of extensive spectroscopic studies, allowing complete stereochemical assignments and subsequently providing a minor reassignment of the side-chain double-bond stereochemistry and, most recently, through total synthesis of authentic material. Here we report the total syntheses of the aglycons of the minor components of the ramoplanin complex, A1 and A3, which unambiguously establish their structure and provide an expected structural revision for the lipid side-chain double-bond stereochemistry.
Mutations of ephrin-B1 (EFNB1), a marker of tissue boundary formation, cause craniofrontonasal syndrome
Authors: Twigg Stephen R. F. , Kan Rui , Babbs Christian , Bochukova Elena G. , Robertson Stephen P. , Wall Steven A. , Morriss-Kay Gillian M. , Wilkie Andrew O. M.
Published: May 2004

Craniofrontonasal syndrome (CFNS) is an X-linked developmental disorder that shows paradoxically greater severity in heterozygous females than in hemizygous males. Females have frontonasal dysplasia and coronal craniosynostosis (fusion of the coronal sutures); in males, hypertelorism is the only typical manifestation. Here, we show that the classical female CFNS phenotype is caused by heterozygous loss-of-function mutations inEFNB1, which encodes a member of the ephrin family of transmembrane ligands for Eph receptor tyrosine kinases. In mice, the orthologousEfnb1gene is expressed in the frontonasal neural crest and demarcates the position of the future coronal suture. AlthoughEFNB1is X-inactivated, we did not observe markedly skewed X-inactivation in either blood or cranial periosteum from females with CFNS, indicating that lack of ephrin-B1 does not compromise cell viability in these tissues. We propose that in heterozygous females, patchwork loss of ephrin-B1 disturbs tissue boundary formation at the developing coronal suture, whereas in males deficient in ephrin-B1, an alternative mechanism maintains the normal boundary. This is the only known mutation in the ephrin/Eph receptor signaling system in humans and provides clues to the biogenesis of craniosynostosis.
O-linked glycoproteins of the nuclear pore complex interact with a cytosolic factor required for nuclear protein import.
Authors: Sterne-Marr R , Blevitt J M , Gerace L
Published: May 2004

Mediated import of proteins into the nucleus requires cytosolic factors and can be blocked by reagents that bind to O-linked glycoproteins of the nuclear pore complex. To investigate whether a cytosolic transport factor directly interacts with these glycoproteins, O-linked glycoproteins from rat liver nuclear envelopes were immobilized on Sepharose beads via wheat germ agglutinin or specific antibodies. When rabbit reticulocyte lysate (which provides cytosolic factors required for in vitro nuclear import) was incubated with the immobilized glycoproteins, the cytosol was found to be inactivated by up to 80% in its ability to support mediated protein import in permeabilized mammalian cells. Inactivation of the import capacity of cytosol, which was specifically attributable to the glycoproteins, involves stoichiometric interactions and is likely to involve binding and depletion of a required factor from the cytosol. This factor is distinct from an N-ethylmaleimide-sensitive receptor for nuclear localization sequences characterized recently since it is insensitive to N-ethylmaleimide. Cytosol inactivation is suggested to be caused by at least two proteins of the glycoprotein fraction, although substantial capacity for inactivation can be attributed to protein bound by the RL11 antibody, consisting predominantly of a 180-kD glycosylated polypeptide. Considered together, these experiments identify a novel cytosolic factor required for nuclear protein import that directly interacts with O-linked glycoproteins of the pore complex, and provide a specific assay for isolation of this component.
GTP hydrolysis is required for vesicle fusion during nuclear envelope assembly in vitro.
Authors: Boman A L , Delannoy M R , Wilson K L
Published: May 2004

Nuclear envelope assembly was studied in vitro using extracts from Xenopus eggs. Nuclear-specific vesicles bound to demembranated sperm chromatin but did not fuse in the absence of cytosol. Addition of cytosol stimulated vesicle fusion, pore complex assembly, and eventual nuclear envelope growth. Vesicle binding and fusion were assayed by light and electron microscopy. Addition of ATP and GTP to bound vesicles caused limited vesicle fusion, but enclosure of the chromatin was not observed. This result suggested that nondialyzable soluble components were required for nuclear vesicle fusion. GTP gamma S and guanylyl imidodiphosphate significantly inhibited vesicle fusion but had no effect on vesicle binding to chromatin. Preincubation of membranes with 1 mM GTP gamma S or GTP did not impair vesicle binding or fusion when assayed with fresh cytosol. However, preincubation of membranes with GTP gamma S plus cytosol caused irreversible inhibition of fusion. The soluble factor mediating the inhibition by GTP gamma S, which we named GTP-dependent soluble factor (GSF), was titratable and was depleted from cytosol by incubation with excess membranes plus GTP gamma S, suggesting a stoichiometric interaction between GSF and a membrane component in the presence of GTP gamma S. In preliminary experiments, cytosol depleted of GSF remained active for fusion of chromatin-bound vesicles, suggesting that GSF may not be required for the fusion reaction itself. We propose that GTP hydrolysis is required at a step before the fusion of nuclear vesicles.
Characterization of the membrane binding and fusion events during nuclear envelope assembly using purified components.
Authors: Newport J , Dunphy W
Published: May 2004

At the end of mitosis membrane vesicles are targeted to the surface of chromatin and fuse to form a continuous nuclear envelope. To investigate the molecular mechanisms underlying these steps in nuclear envelope assembly, we have developed a defined cell-free system in which the binding and fusion steps in nuclear envelope assembly can be examined separately. We have found that extensively boiled Xenopus egg extracts efficiently promote the decondensation of demembranated Xenopus sperm chromatin. When isolated membranes are added to this decondensed chromatin a specific subfraction of membrane vesicles (approximately 70 nM in diameter) bind to the chromatin, but these vesicles do not fuse to each other. Vesicle binding is independent of ATP and insensitive to N-ethylmalamide. Quantitative analysis of these sites by EM suggests that there is at least one vesicle binding site per 100 kb of chromosomal DNA. We show by tryptic digestion that vesicle-chromatin association requires proteins on both the vesicle and on the chromatin. In addition, we show that the vesicles bound under these conditions will fuse into an intact nuclear envelope when incubated with the soluble fraction of a Xenopus egg nuclear assembly extract. With respect to vesicle fusion, we have found that vesicles prebound to chromatin will fuse to each other when ATP and GTP are present in the boiled extract. These results indicate that nuclear envelope assembly is mediated by a subset of approximately 70-nM-diam vesicles which bind to chromatin sites spaced 100 kb apart and that fusion of these vesicles is regulated by membrane-associated GTP-binding proteins.
Association of the tyrosine phosphorylated epidermal growth factor receptor with a 55-kD tyrosine phosphorylated protein at the cell surface and in endosomes.
Authors: Wada I , Lai W H , Posner B I , Bergeron J J
Published: May 2004

After the intraportal injection of EGF, the EGF receptor (EGFR) is rapidly internalized into hepatic endosomes where it remains largely receptor bound (Lai et al., 1989. J. Cell Biol. 109:2751-2760). In the present study, we evaluated the phosphotyrosine content of EGFRs at the cell surface and in endosomes in order to assess the consequences of internalization. Quantitative estimates of specific radioactivity of the EGFR in these two compartments revealed that tyrosine phosphorylation of the EGFR was observed at the cell surface within 30 s of ligand administration. However, the EGFR was also highly phosphorylated in endosomes reaching levels of tyrosine phosphorylation significantly higher than those of the cell surface receptor at 5 and 15 min after EGF injection. A 55-kD tyrosine phosphorylated polypeptide (pyp55) was observed in association with the EGFR at the cell surface within 30 s of EGF injection. The protein was also found in association with the EGFR in endosomes as evidenced by coprecipitation studies using a mAb to the EGFR as well as by coelution with the EGR in gel permeation chromatography. Limited proteolysis of isolated endosomes indicated that the tyrosine phosphorylated domains of the EGFR and associated pyp55 were cytosolically oriented while internalized EGF was intraluminal. The identification of pyp55 in association with EGFR in both hepatic plasma membranes and endosomes may be relevant to EGFR function and/or trafficking of the EGFR.
Cell-free fusion of endocytic vesicles is regulated by phosphorylation.
Authors: Woodman P G , Mundy D I , Cohen P , Warren G
Published: May 2004

Okadaic acid and microcystin-LR, both potent inhibitors of protein phosphatases (PP), blocked vesicle fusion in a cell-free system. The effect of okadaic acid was reversed by the purified catalytic subunit of PP2A, but not PP1. Inhibition was gradual, required Mg-ATP, and was reduced by protein kinase inhibitors, indicating that it was mediated via protein phosphorylation. A candidate protein kinase would be cdc2 kinase, which normally is active in mitotic extracts and has been shown to inhibit endocytic vesicle fusion (Tuomikoski, T., M.-A. Felix, M. Dorée, and J. Gruenberg. 1989. Nature (Lond.). 342:942-945). However, it would appear that cdc2 kinase is not responsible for inhibition by okadaic acid. When compared to cytosol prepared from mitotic cells, okadaic acid did not increase cdc2 kinase activity sufficiently to account for the inhibition. In addition, inhibition was maintained when cdc2 protein was depleted from cytosol.
Membrane fusion process of Semliki Forest virus. I: Low pH-induced rearrangement in spike protein quaternary structure precedes virus penetration into cells.
Authors: Wahlberg J M , Garoff H
Published: May 2004

The Semliki Forest virus (SFV) directs the synthesis of a heterodimeric membrane protein complex which is used for virus membrane assembly during budding at the surface of the infected cell, as well as for low pH-induced membrane fusion in the endosomes when particles enter new host cells. Existing evidence suggests that the E1 protein subunit carries the fusion potential of the heterodimer, whereas the E2 subunit, or its intracellular precursor p62, is required for binding to the nucleocapsid. We show here that during virus uptake into acidic endosomes the original E2E1 heterodimer is destabilized and the E1 proteins form new oligomers, presumably homooligomers, with altered E1 structure. This altered structure of E1 is specifically recognized by a monoclonal antibody which can also inhibit penetration of SFV into host cells as well as SFV-mediated cell-cell fusion, thus suggesting that the altered E1 structure is important for the membrane fusion. These results give further support for a membrane protein oligomerization-mediated control mechanism for the membrane fusion potential in alphaviruses.
Membrane fusion process of Semliki Forest virus. II: Cleavage-dependent reorganization of the spike protein complex controls virus entry.
Authors: Salminen A , Wahlberg J M , Lobigs M , Liljeström P , Garoff H
Published: May 2004

The envelope of the Semliki Forest virus (SFV) contains two transmembrane proteins, E2 and E1, in a heterodimeric complex. The E2 subunit is initially synthesized as a precursor protein p62, which is proteolytically processed to the mature E2 form before virus budding at the plasma membrane. The p62 (E2) protein mediates binding of the heterodimer to the nucleocapsid during virus budding, whereas E1 carries the entry functions of the virus, that is, cell binding and low pH-mediated membrane fusion activity. We have investigated the significance of the cleavage event for the maturation and entry of the virus. To express SFV with an uncleaved p62 phenotype, BHK-21 cells were transfected by electroporation with infectious viral RNA transcribed from a full-length SFV cDNA clone in which the p62 cleavage site had been changed. The uncleaved p62E1 heterodimer was found to be used for the formation of virus particles with an efficiency comparable to the wild type E2E1 form. However, in contrast to the wild type virus, the mutant virus was virtually noninfectious. Noninfectivity resulted from impaired uptake into cells, as well as from the inability of the virus to promote membrane fusion in the mildly acidic conditions of the endosome. This inability could be reversed by mild trypsin treatment, which converted the viral p62E1 form into the mature E2E1 form, or by treating the virus with a pH 4.5 wash, which in contrast to the more mild pH conditions of endosomes, effectively disrupted the p62E1 subunit association. We conclude that the p62 cleavage is not needed for virus budding, but regulates entry functions of the E1 subunit by controlling the heterodimer stability in acidic conditions.
Genetic analysis of a Drosophila microtubule-associated protein.
Authors: Pereira A , Doshen J , Tanaka E , Goldstein L S
Published: May 2004

The 205-kD microtubule-associated protein (205K MAP) is one of the principal MAPs in Drosophila. 205K MAP is similar to the HeLa 210K/MAP4 family of MAPs since it shares the following biochemical properties: it is present in several isoforms, has a molecular mass of approximately 200 kD, and is thermostable. Furthermore, immuno-crossreactivity has been observed between mouse MAP4, HeLa 210K, and Drosophila 205K MAP. Currently, there is little information concerning the biological function of this group of nonmotor MAPs. We have used a classical genetic approach to try to identify the role of the 205K MAP in Drosophila by isolating mutations in the 205K MAP gene. An F2 lethal screen was used to acquire deficiencies of 100EF, the chromosomal location of the 205K MAP gene. Drosophila bearing a homozygous deficiency for the 205K MAP region are fully viable and show no obvious phenotype. A recently developed polymerase chain reaction screen was also used to recover five P-element insertions upstream from the 205K MAP gene. Western blot analysis has shown that these insertions result in hypomorphic mutations of the 205K MAP gene. As was seen with animals that have no 205K MAP, these mutations appear to have no phenotype. These data unambiguously demonstrate that the 205K MAP gene is inessential for development. These results also suggest that there may exist protein(s) with redundant function that can substitute for 205K MAP.
Expression of fusion proteins of the nicotinic acetylcholine receptor from mammalian muscle identifies the membrane-spanning regions in the alpha and delta subunits.
Authors: Chavez R A , Hall Z W
Published: May 2004

We have investigated the topology of the alpha and delta subunits of the nicotinic acetylcholine receptor (AChR) from mammalian muscle synthesized in an in vitro translation system supplemented with dog pancreatic microsomes. Fusion proteins were expressed in which a carboxy-terminal fragment of bovine prolactin was attached downstream of each of the major putative transmembrane domains, M1-M4 and MA, in the AChR subunits. The orientation of the prolactin domain relative to the microsomal membrane was then determined for each protein by a proteolysis protection assay. Since the prolactin domain contains no information which either directs or prevents its translocation, its transmembrane orientation depends solely on sequences within the AChR subunit portion of the fusion protein. When subunit-prolactin fusion proteins with the prolactin domain fused after either M2 or M4 were tested, prolactin-immunoreactive peptides that were larger than the prolactin domain itself were recovered. No prolactin-immunoreactive peptides were recovered after proteolysis of fusion proteins containing prolactin fused after M1, M3, or MA. These results support a model of AChR subunit topology in which M1-M4, but not MA, are transmembrane domains and the carboxy terminus is extracellular.
Myelin sheath survival after guanethidine-induced axonal degeneration.
Authors: Kidd G J , Heath J W , Trapp B D , Dunkley P R
Published: May 2004

Membrane-membrane interactions between axons and Schwann cells are required for initial myelin formation in the peripheral nervous system. However, recent studies of double myelination in sympathetic nerve have indicated that myelin sheaths continue to exist after complete loss of axonal contact (Kidd, G. J., and J. W. Heath. 1988. J. Neurocytol. 17:245-261). This suggests that myelin maintenance may be regulated either by diffusible axonal factors or by nonaxonal mechanisms. To test these hypotheses, axons involved in double myelination in the rat superior cervical ganglion were destroyed by chronic guanethidine treatment. Guanethidine-induced sympathectomy resulted in a Wallerian-like pattern of myelin degeneration within 10 d. In doubly myelinated configurations the axon, inner myelin sheath (which lies in contact with the axon), and approximately 75% of outer myelin sheaths broke down by this time. Degenerating outer sheaths were not found at later periods. It is probably that outer sheaths that degenerated were only partially displaced from the axon at the commencement of guanethidine treatment. In contrast, analysis of serial sections showed that completely displaced outer internodes remained ultrastructurally intact. These internodes survived degeneration of the axon and inner sheath, and during the later time points (2-6 wk) they enclosed only connective tissue elements and reorganized Schwann cells/processes. Axonal regeneration was not observed within surviving outer internodes. We therefore conclude that myelin maintenance in the superior cervical ganglion is not dependent on direct axonal contact or diffusible axonal factors. In addition, physical association of Schwann cells with the degenerating axon may be an important factor in precipitating myelin breakdown during Wallerian degeneration.
A strategy for isolation of cDNAs encoding proteins affecting human intestinal epithelial cell growth and differentiation: characterization of a novel gut-specific N-myristoylated annexin.
Authors: Wice B M , Gordon J I
Published: May 2004

The human intestinal epithelium is rapidly and perpetually renewed as the descendants of multipotent stem cells located in crypts undergo proliferation, differentiation, and eventual exfoliation during a very well organized migration along the crypt to villus axis. The mechanisms that establish and maintain this balance between proliferation and differentiation are largely unknown. We have utilized HT-29 cells, derived from a human colon adenocarcinoma, as a model system for identifying gene products that may regulate these processes. Proliferating HT-29 cells cultured in the absence of glucose (e.g., using inosine as the carbon source) have some of the characteristics of undifferentiated but committed crypt epithelial cells while postconfluent cells cultured in the absence of glucose resemble terminally differentiated enterocytes or goblet cells. A cDNA library, constructed from exponentially growing HT-29 cells maintained in inosine-containing media, was sequentially screened with a series of probes depleted of sequences encoding housekeeping functions and enriched for intestine-specific sequences that are expressed in proliferating committed, but not differentiated, epithelial cells. Of 100,000 recombinant phage surveyed, one was found whose cDNA was derived from an apparently gut-specific mRNA. It encodes a 316 residue, 35,463-D protein that is a new member of the annexin/lipocortin family. Other family members have been implicated in regulation of cellular growth and in signal transduction pathways. RNA blot and in situ hybridization studies indicate that the gene encoding this new annexin exhibits region-specific expression along both axes of the human gut: (a) highest levels of mRNA are present in the jejunum with marked and progressive reductions occurring distally; (b) its mRNA appears in crypt-associated epithelial cells and increases in concentration as they exit the crypt. Villus-associated epithelial cells continue to transcribe this gene during their differentiation/translocation up the villus. Immunocytochemical studies reveal that the intestine-specific annexin (ISA) is associated with the plasma membrane of undifferentiated, proliferating crypt epithelial cells as well as differentiated villus enterocytes. In polarized enterocytes, the highest concentrations of ISA are found at the apical compared to basolateral membrane. In vitro studies using an octapeptide derived from residues 2-9 of the primary translation product of ISA mRNA and purified myristoyl-CoA:protein N-myristoyltransferase suggested that it is N-myristoylated. In vivo labeling studies confirmed that myristate is covalently attached to ISA via a hydroxylamine resistant amide linkage. The restricted cellular expression and acylation of ISA distinguish it from other known annexins.(ABSTRACT TRUNCATED AT 400 WORDS)
Phorbol ester induced osteoclast-like differentiation of a novel human leukemic cell line (FLG 29.1).
Authors: Gattei V , Bernabei P A , Pinto A , Bezzini R , Ringressi A , Formigli L , Tanini A , Attadia V , Brandi M L
Published: May 2004

Studies on human osteoclast formation have been hampered by lack of a defined isolated progenitor cell population. We describe here the establishment of a human leukemic cell line (designated FLG 29.1) from bone marrow of a patient with acute monoblastic leukemia. The cultured cells are predominantly undifferentiated leukemic blasts, but addition of 12-o-tetradecanoylphorbol 13-acetate (TPA; 0.1 microM) induces irreversible differentiation into adherent, non-dividing, multinucleated cells. TPA-treated cells bear surface antigens typical of fetal osteoclasts, degrade 45Ca-labeled devitalized bone particles, display tartrate-resistant acid phosphatase in both mononuclear and multinuclear cells and receptors for calcitonin. Calcitonin increases intracellular cAMP accumulation in TPA-treated cells. TPA-treated cells show some ultrastructural features of osteoclasts as evidenced by transmission EM. These results indicate that FLG 29.1 cells may represent an osteoclast committed cell population, which upon induction with TPA acquire some morphological, phenotypical, and functional features of differentiated osteoclasts.
Clusters of neural cell adhesion molecule at sites of cell-cell contact.
Authors: Bloch R J
Published: May 2004

I have examined the distribution of neural cell adhesion molecule (N-CAM) in cultured C2 myogenic cells and other cell lines to determine if N-CAM accumulates at sites of cell-cell contact. C2 cells growing in log phase display large clusters of neural cell adhesion molecule where they contact each other. These clusters are remarkably stable, do not form at cell-substrate contacts, and appear not to be enriched in a number of other cytoskeletal, membrane, or extracellular proteins. Thus, N-CAM clusters form preferentially in response to cell-cell contact and are specifically enriched in N-CAM. As C2 cultures mature and differentiate, clusters persist at contacts between aligning myoblasts and between myotubes, consistent with a role in myogenesis. N-CAM is also enriched at cell-cell contacts in cultures of PC12, NRK, and CHO cells. These cells have significant amounts of N-CAM as detected on immunoblots. Clusters are not seen in L929 cells, which do not have detectable amounts of N-CAM. Coculture of these cells with C2 cells results in the clustering of N-CAM at heterologous contacts between C2 cells and NRK, CHO, or PC12 cells, but not between C2 cells and L929 cells. These results suggest that N-CAM specifically accumulates where N-CAM-bearing cells contact one another. Clustering of N-CAM may be an important step in strengthening intercellular adhesion.
Inhibition of cell adhesion by high molecular weight kininogen.
Authors: Asakura S , Hurley R W , Skorstengaard K , Ohkubo I , Mosher D F
Published: May 2004

An anti-cell adhesion globulin was purified from human plasma by heparin-affinity chromatography. The purified globulin inhibited spreading of osteosarcoma and melanoma cells on vitronectin, and of endothelial cells, platelets, and mononuclear blood cells on vitronectin or fibrinogen. It did not inhibit cell spreading on fibronectin. The protein had the strongest antiadhesive effect when preadsorbed onto the otherwise adhesive surfaces. Amino acid sequence analysis revealed that the globulin is cleaved (kinin-free) high molecular weight kininogen (HKa). Globulin fractions from normal plasma immunodepleted of high molecular weight kininogen (HK) or from an individual deficient of HK lacked adhesive activity. Uncleaved single-chain HK preadsorbed at neutral pH, HKa preadsorbed at pH greater than 8.0, and HKa degraded further to release its histidine-rich domain had little anti-adhesive activity. These results indicate that the cationic histidine-rich domain is critical for anti-adhesive activity and is somehow mobilized upon cleavage. Vitronectin was not displaced from the surface by HKa. Thus, cleavage of HK by kallikrein results in both release of bradykinin, a potent vasoactive and growth-promoting peptide, and formation of a potent anti-adhesive protein.
Activation-dependent recognition by hematopoietic cells of the LDV sequence in the V region of fibronectin.
Authors: Wayner E A , Kovach N L
Published: May 2004

It has been shown that the alpha 4 beta 1 integrin is the lymphocyte receptor for the carboxy terminal cell-binding domain of fibronectin which comprises adhesion sites in Hep 2 and a high affinity site, CS-1, in the type III connecting segment or V (for variable) region. In the present studies, using a series of peptides derived from CS-1, we identify the tripeptide leu-asp-val (LDV), as the minimal peptide capable of supporting stable lymphocyte or melanoma cell adhesion. However, only cells which expressed an active form of the alpha 4 beta 1 complex were capable of attaching to and spreading on LDV peptide. On a molar basis, LDV minimal peptides were either not active or 10-20 times less active than intact CS-1 in promoting the adhesion of lymphocytes expressing the resting form of the receptor. In cells which express the high avidity form of the receptor, LDV and CS-1 were equally effective in promoting cell adhesion and spreading. The avidity of the alpha 4 beta 1 complex could be altered with mAbs to beta 1 which specifically activate beta 1 dependent function. The high avidity form of the alpha 4 beta 1 complex could be induced on U937 cells, T, and B lymphoblastoid cell lines, or PHA-stimulated T cell blasts. Resting PBL could not be induced to bind LDV peptide conjugates by activating antibodies to beta 1 implying that two signals are required for LDV recognition by T cells. In conclusion, these data show clearly that the minimal peptide for the alpha 4 beta 1 complex in CS-1 is the LDV sequence. Although numerous cell populations can interact with intact CS-1 only cells which express an active alpha 4 beta 1 complex can bind the LDV sequence. This implies that cell interaction with the carboxy terminal cell-binding domain of fibronectin can be regulated at several levels: (a) alpha 4 beta 1 expression; (b) activation of the alpha 4 beta 1 complex; and (c) alternate splicing of CS-1 into V+ isoforms of fibronectin.