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1.
Jonathan A. Lindquist Elisabeth Barofsky Philip N. McFadden 《Journal of Protein Chemistry》1996,15(1):115-122
Protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase (PCM, E.C. 2.1.1.77) was previously shown to be enzymatically methyl esterified in an autocatalytic manner at altered aspartyl residues; methyl esters are observed in a subpopulation of the enzyme termed thePCM fraction [Lindquist and McFadden (1994),J. Protein Chem.
13, 23–30]. The altered aspartyl sites serving as methyl acceptors inPCM have now been localized by using proteolytic enzymes and chemical cleavage techniques in combination with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to identify fragments of the [3H]automethylated enzyme that contain a [3H]methyl ester. Methylation was positively identified at positions Asn188 and Asp217 in the enzyme sequence, a consequence of the spontaneous alteration of these sites tol-isoaspartyl ord-aspartyl sites and their methylation by active PCM molecules. The identification of more than one site of automethylation shows thatPCM is not a homogeneous population of damaged PCM molecules, but rather a complex population of molecules with a variety of age-altered damage sites.Abbreviations PCM
protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase
- EDTA
disodium ethylenediaminetetraacetate
- PMSF
phenylmethylsulfonyl fluoride
- TEA
trifluoroacetic acid
- HPLC
high-pressure liquid chromatography 相似文献
2.
Calcium content and distribution as a function of growth and transformation in the mouse 3T3 cell 下载免费PDF全文
Total Ca content and that fraction of Ca sensitive to removal by the chelator ethylene glycol-bis(β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) have been investigated in the mouse 3T3 cell as a function of growth stage, transformation with SV40 virus, and serum levels of the media. Cells were allowed to grow through several doublings in media containing (45)Ca. The cellular content of (45)Ca was used to access total cell Ca. That fraction of (45)Ca removed by EGTA was presumed to represent primarily surface-localized Ca. The data are expressed on a per cell volume basis to compensate for size differences as a function of growth stage and transformation. During exponential growth phase, the 3T3 cell contains 525pmol Ca/μl cell volume. Of this, approx. 457 pmol/μl is not removable by EGTA and, presumably, is cytoplasmically located. This value is in close agreement with previous studies on the HeLa cell (470 pmol Ca/μl cell water after the removal of the surface Ca). The low level of EGTA- removable Ca present in the 3T3 cell during early exponential growth (68 pmol Ca/μl cell volume) increases progressively with increasing cell density, and upon quiescence it is sevenfold greater. In contrast, SV40- transformed 3T3 cells growing exponentially possess total levels of Ca which are approximately two-thirds the levels of the normal 3T3 cell. However, their EGTA-sensitive Ca is not significantly different from that of exponentially growing, normal 3T3 cells. As the transformed cells continue to grow at high density, their total ca and their sensitivity to EGTA do not change, in contrast to the normal 3T3 cell. Thus, an increase in Ca associated with the cell surface appears to be correlated with growth inhibition. This has been investigated further by regulating growth of the normal and transformed cell with alterations in the serum level of the media. In 4 percent calf serum the normal cell is stopped from continued proliferation. Growth stoppage under these conditions is characterized by a nearly fourfold increase in EGTA-removable Ca, similar to the increase observed upon quiescence in depleted 10 percent serum. Similar treatment of the transformed cell does not reduce its growth rate, nor does it significantly alter Ca distribution. However, at 0.5 percent medium serum levels, the SV40 3T3 growth rate is substantially reduced and, under these conditions, EGTA-removable Ca increases twofold. 相似文献
3.
William r. Anderson Willi Frick G.Doyle Daves Douglas F. Barofsky Isomaro Yamaguchi Ding Chang Karl Folkers Sune Rosell 《Biochemical and biophysical research communications》1977,78(1):372-376
Mass spectra of underivatized hexa- and heptapeptide amides related to Substance P have been obtained with a conventional electron ionization mass spectrometer using sample vaporization from a tungsten wire by the technique of rapid heating, proton transfer ionization using ammonia, and photoplate recording of spectra. These spectra exhibit little evidence of sample pyrolysis and are readily interpreted to yield amino acid sequences. 相似文献
4.
Lai CM Yu MJ Brankov M Barnett NL Zhou X Redmond TM Narfstrom K Rakoczy PE 《Genetic vaccines and therapy》2004,2(1):3
Background
Leber's congenital amaurosis (LCA) is a severe form of retinal dystrophy. Mutations in the RPE65 gene, which is abundantly expressed in retinal pigment epithelial (RPE) cells, account for approximately 10–15% of LCA cases. In this study we used the high turnover, and rapid breeding and maturation time of the Rpe65 -/- knockout mice to assess the efficacy of using rAAV-mediated gene therapy to replace the disrupted RPE65 gene. The potential for rAAV-mediated gene treatment of LCA was then analyzed by determining the pattern of RPE65 expression, the physiological and histological effects that it produced, and any improvement in visual function. 相似文献5.
Background
Long-lasting insecticidal nets (LLINs) reduce malaria transmission by protecting individuals from infectious bites, and by reducing mosquito survival. In recent years, millions of LLINs have been distributed across sub-Saharan Africa (SSA). Over time, LLINs decay physically and chemically and are destroyed, making repeated interventions necessary to prevent a resurgence of malaria. Because its effects on transmission are important (more so than the effects of individual protection), estimates of the lifetime of mass distribution rounds should be based on the effective length of epidemiological protection.Methods
Simulation models, parameterised using available field data, were used to analyse how the distribution's effective lifetime depends on the transmission setting and on LLIN characteristics. Factors considered were the pre-intervention transmission level, initial coverage, net attrition, and both physical and chemical decay. An ensemble of 14 stochastic individual-based model variants for malaria in humans was used, combined with a deterministic model for malaria in mosquitoes.Results
The effective lifetime was most sensitive to the pre-intervention transmission level, with a lifetime of almost 10 years at an entomological inoculation rate of two infectious bites per adult per annum (ibpapa), but of little more than 2 years at 256 ibpapa. The LLIN attrition rate and the insecticide decay rate were the next most important parameters. The lifetime was surprisingly insensitive to physical decay parameters, but this could change as physical integrity gains importance with the emergence and spread of pyrethroid resistance.Conclusions
The strong dependency of the effective lifetime on the pre-intervention transmission level indicated that the required distribution frequency may vary more with the local entomological situation than with LLIN quality or the characteristics of the distribution system. This highlights the need for malaria monitoring both before and during intervention programmes, particularly since there are likely to be strong variations between years and over short distances. The majority of SSA's population falls into exposure categories where the lifetime is relatively long, but because exposure estimates are highly uncertain, it is necessary to consider subsequent interventions before the end of the expected effective lifetime based on an imprecise transmission measure. 相似文献6.
Marcos G Godoy Alejandra Aedo Molly JT Kibenge David B Groman Carmencita V Yason Horts Grothusen Angelica Lisperguer Marlene Calbucura Fernando Avendaño Marcelo Imilán Miguel Jarpa Frederick SB Kibenge 《BMC veterinary research》2008,4(1):1-13
Background
Several forms of progressive retinal atrophy (PRA) segregate in more than 100 breeds of dog with each PRA segregating in one or a few breeds. This breed specificity may be accounted for by founder effects and genetic drift, which have reduced the genetic heterogeneity of each breed, thereby facilitating the identification of causal mutations. We report here a new form of PRA segregating in the Border Collie breed. The clinical signs, including the loss of night vision and a progressive loss of day vision, resulting in complete blindness, occur at the age of three to four years and may be detected earlier through systematic ocular fundus examination and electroretinography (ERG). 相似文献7.
A cultured rat ovarian cell line (31 A-F(2)) was used to study the effect of growth factors (epidermal growth factor [EGF] and fibroblast growth factor [FGF]), a survival factor (ovarian growth factor [OGF]), a hormone (insulin), and an iron-binding protein (transferring) on cell proliferation and steroid production under defined culture conditions. EGF and insulin were shown to be mitogenic (half-maximal response at 0.12 nM and 0.11 muM, respectively) for 31A-F(2) cells incubated in serum-free medium. EGF induced up to three doublings in the cell population, whereas insulin induced an average of one cell population doubling. FGF, OGF, and transferrin were found not to have any prominent effect on cell division when incubated individually with 31A-F(2) cells in serum-free medium. However, a combination of EGF, OGF, insulin, and transferrin stimulated cell division to the same approximate extent as cells incubated in the presence of 5 percent fetal calf serum. EGF or insulin did not significantly affect total cell cholesterol levels (relative to cells incubated in serum-free medium) when incubated individually with 31A-F(2) cells. However, cell cholesterol levels were increased by the addition of OGF (250 percent), FGF (370 percent), or a combination of insulin and EGF (320 percent). Progesterone secretion from 31A-F(2) cells was enhanced by EGF (25 percent), FGF (80 percent), and insulin (115 percent). However, the addition of a mitogenic mixture of EGF, OGF, insulin, and transferrin suppressed progesterone secretion 150 percent) below that of control cultures. These studies have permitted us to determine that EGF and insulin are mitogenic factors that are required for the growth of 31A-F(2) cells and that OGF and transferrin are positive cofactors that enhance growth. Also, additional data suggest that cholesterol and progesterone production in 31A-F(2) cells can be regulated by peptide growth factors and the hormone insulin. 相似文献
8.
Gafken PR Doneanu CE Bennett SE Barofsky DF 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2007,860(2):145-152
In this report, the effectiveness of high performance liquid chromatography (HPLC) in conjunction with electrospray ionization mass spectrometry (ESI-MS) is examined as a tool for identifying the sites of crosslinking in a protein that has been photoreacted with a non-photolabeled oligonucleotide. ESI-MS and MALDI-MS analyses preceded by off-line microflow and nanoflow HPLC, on-line microflow HPLC/ESI, and on-line nanoflow HPLC/ESI interfaces were performed in order to determine their relative effectiveness in separating mixtures of nucleopeptides and identifying sites of crosslinking on the individual components. The characteristics of these four techniques as well as possibilities for improving the analysis of nucleopeptides by ESI-MS are compared and discussed. 相似文献
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10.
Frederick SB Kibenge Marcos G Godoy Yingwei Wang Molly JT Kibenge Valentina Gherardelli Soledad Mansilla Angelica Lisperger Miguel Jarpa Geraldine Larroquete Fernando Avendaño Marcela Lara Alicia Gallardo 《Virology journal》2009,6(1):1-16