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81.
A coupling constant-dihedral angle correlation for the H? Cα? Cβ? H system of amino acid residues in peptides has been derived from a set of model compounds covering the full range of dihedral angles. The expression obtained, J = 11.0 cos2 θ ?1.4 cos θ + 1.6 sin2θ, is close to those already used in pmr studies of peptide conformation, and provides a firmer foundation for them. A factor limiting the precision of this and other “Karplus relations” is illustrated. 相似文献
82.
An Improved Diluent for Rubella Hemagglutination and Hemagglutination-Inhibition Tests 总被引:3,自引:2,他引:1 下载免费PDF全文
Angela E. Auletta Gary L. Gitnick Carrie E. Whitmire John L. Sever 《Applied microbiology》1968,16(5):691-694
Rubella hemagglutinating (HA) antigen was prepared in BHK-21 tissue as 5% cell suspensions and from unconcentrated and 20× concentrated infected supernatant fluids. In some instances, unconcentrated fluids were treated with Tween 80 and ether; cell suspensions were treated with ether alone. Preparations were tested for HA activity in dextrose-gelatin-Veronal (DGV) buffer solutions; 0.85% NaCl; Sorenson's phosphate-buffered saline, pH 7.2; and a diluent of 0.9% NaCl, 0.1% CaCl2 (anhydrous), and 0.1% MgSO4·7H2O. HA titers were consistently two- to fourfold higher in the saline with added Ca++ and Mg++ than in DGV. Hemagglutination-inhibition titers of paired human sera were the same in either diluent. It is suggested that the interaction between rubella HA antigen and the red cells of young (less than 1-day-old) chicks may be at least partially ion dependent and that titers are enhanced by increased quantities of divalent cations. 相似文献
83.
Tarek Bisat Terry R. Brown Claude J. Migeon Gary D. Berkovitz 《In vitro cellular & developmental biology. Plant》1989,25(9):806-812
Summary Because the measurement of aromatase activity in cultured human genital skin fibroblasts has been proposed as a means of studying
estrogen production in men, we investigated the influence of culture conditions on aromatase activity.
Genital skin fibroblasts were seeded onto culture plates at a density of 1×106 cells/plate and aromatase activity was determined over a 1-mo. period. Enzyme activity rose slowly over the first 14 d but
then rose rapidly to a 10-fold higher plateau by Day 28. The rise in aromatase activity was similar whether activity was normalized
for protein or for DNA content. When cells were seeded at the usual density of 1×106 or at 0.25×106 cells/plate, aromatase activity was consistently lower during the first 2 wk in cells plated at lower density, but thereafter
the levels of enzyme activity in the two groups converged. In cells plated at the lower density, the lower activity observed
in the first 2 wk was associated with a lower V
max
. Preincubation of cells plated at one density with conditoned medium from cells plated at the other density did not change
the relatve levels of activity in the two groups. By contrast, dihydrotestosterone (DHT) receptor binding and 5α-reductase
activity were similar at all time points, despite differences in plating density.
In additional experiments, the culture medium was replaced daily rather than every 3rd d, and aromatase activity was assayed
on Day 7. In cells fed daily, DNA and protein content were twice that of cells fed every 3rd d. By contrast, aromatase activity
declined to 30% of the in the latter group. DHT and dexamethasone receptor binding and 5α-reductase activity were similar
in the two groups.
In summary, factors such as plating density, culture density, and frequency of media replacement dramatically influence aromatase
activity in cultured human genital skin fibroblasts. Therefore, the interpretation of aromatase activity data obtained from
cultured cells in relation to physiologic or pathologic states should be viewed with appropriate caution.
The work was supported in part by grants R01 DK 35339 and R01 DK 00180 from the National Institutes of Health, Bethesda, MD,
and by RR 00035 from CLINFO Systems at the Johns Hopkins University School of Medicine, Baltimore, MD. 相似文献
84.
Treatment differences were observed in the in vitro adventitious shoot regeneration response from internodal explants of three genotypes of Populus deltoides cultured on media supplemented with five concentrations each of the cytokinins 6-benzyladenine, 2-isopentyladenine, and zeatin. For each of the three genotypes, the greatest number of shoots were consistently regenerated on media containing the cytokinin zeatin. Tissue necrosis resulted when explants from any of the three genotypes were cultured on media supplemented with 6-benzyladenine. A zeatin concentration by genotype interaction was also observed. Genotypic differences in shoot regeneration were observed for 16 genotypes of Populus deltoides when cultured on medium supplemented with 0.5 mgL–1 zeatin. Six genotypes were highly recalcitrant and failed to regenerate shoots. The percent of explants regenerating was greater than 50% for four genotypes.Abbreviations WNA
modified Woody Plant Media
- BA
N6-benzyladenine
- 2-iP
2-isopentenyladenine
- 2,4-D
2,4-dichlorophenoxyacetic acid
- IBA
indole-3-butyric acid
- MS
Murashige Skoog (1962) medium
- NAA
1-naphthaleneacetic acid
- PAR
photosynthetically active radiation
Journal Series No. 8938, Agricultural Research Division, University of Nebraska 相似文献
85.
Characterization of a Nerve Growth Factor-Stimulated Protein Kinase in PC12 Cells Which Phosphorylates Microtubule-Associated Protein 2 and pp250 总被引:15,自引:5,他引:10
Gary E. Landreth Deanna S. Smith Craig McCabe Cynthia Gittinger 《Journal of neurochemistry》1990,55(2):514-523
Treatment of PC12 cells with nerve growth factor (NGF) resulted in the rapid, but transient, activation of a protein kinase which specifically phosphorylated an endogenous 250-kDa cytoskeletal protein (pp250). We report that the microtubule-associated protein, MAP2, is an alternative substrate for the NGF-activated kinase. NGF treatment maximally activated the kinase within 5 min; however, the activity declined with longer exposure to NGF. The enzyme was localized predominantly in microsomal and soluble fractions and phosphorylated MAP2 on serine and threonine residues. The soluble enzyme was fractionated by DEAE chromatography and gel filtration and had an apparent Mr of 45,000. The enzyme was purified to near homogeneity by chromatofocussing and had a pI of 4.9. Kinetic analysis revealed that NGF treatment caused a sevenfold increase in Vmax for MAP2. The Km with respect to the MAP2 substrate was approximately 50 nM and was not altered by NGF treatment. A novel feature of the NGF-stimulated enzyme was its sharp dependence on Mn2+ concentration. The active enzyme is likely to be phosphorylated, because inclusion of phosphatase inhibitors was required for recovery of optimal activity and the activity was lost on treatment of the enzyme with alkaline phosphatase. Histones, tubulin, casein, bovine serum albumin, and the ribosomal subunit protein S-6 were not phosphorylated by this enzyme. The NGF-stimulated kinase was distinct from A kinase, C kinase, or other NGF-stimulated kinases. The rapid and transient activation of the protein kinase upon NGF treatment suggests that the enzyme may play a role in signal transduction in PC12 cells. 相似文献
86.
Transport of Tryptophan into Brain from the Circulating, Albumin-Bound Pool in Rats and in Rabbits 总被引:2,自引:2,他引:0
Tryptophan is the only amino acid in the circulation that is bound by albumin, and previous studies have suggested that the brain tryptophan supply is a function of either the free or the albumin-bound pool of tryptophan in blood. Since the albumin molecule per se does not cross the brain capillary wall, i.e., the blood-brain barrier (BBB), the transport of tryptophan from the circulating albumin-bound pool may involve enhanced dissociation of tryptophan from the albumin binding sites within the cerebral microcirculation. This hypothesis was confirmed in the present studies wherein the dissociation constant (KaD) of albumin binding of tryptophan in the rat or rabbit brain microcirculation was measured in vivo. Brain extraction data for [14C]tryptophan determined with the carotid artery injection technique were fit to the Kety-Renkin-Crone equation modified for protein-bound solute. The KaD of albumin binding in the rat or rabbit brain microcirculation under pentobarbital anesthesia was 1.7 +/- 0.1 and 3.9 +/- 1.0 mM, respectively, as compared to the KD value measured in vitro with equilibrium dialysis, 0.13 +/- 0.03 mM. In contrast, the KaD value of albumin binding of tryptophan in vivo in the rabbit brain microcirculation was reduced by ether anesthesia to a value of 2.1 +/- 0.4 mM. This reduction in the KaD under ether anesthesia was associated with a 2.5-fold increase in cerebral blood flow. In addition, dialyzed rabbit serum caused a statistically significant inhibition in [14C]tryptophan influx during ether, but not pentobarbital, anesthesia.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
87.
Gary M. Stuhlmiller Kathryn M. Roberson H. F. Seigler 《Cancer immunology, immunotherapy : CII》1989,29(3):205-210
Summary The immunogenicity of the disialoganglioside, GD3, a melanoma-tumor-associated antigen, has been evaluated in non-human primates. Sera from four chimpanzees and two monkeys were evaluated for anti-GD3 antibody activity by solid-phase radioimmunoassay using GD3 and control gangliosides as targets. Serum from one monkey, immunized with cells from a melanoma cell line, was strongly reactive with GD3, having a titer of >2500. In contrast, serum from this animal was non-reactive with several other gangliosides including the structurally similar GM3. Anti-GD3 reactivity was also demonstrable, albeit in low titer, in the sera of an additional monkey and a chimpanzee. Each of these animals had likewise been immunized using cells from melanoma cell lines. On the basis of these observations, suggestive of a primate anti-GD3 antibody response, we initiated a series of immunizations of chimpanzee using purified GD3 bound to Salmonella minnesota, R595. IgG reactive with melanoma cells in the cell-binding assay was first detected in sera collected after 4 immunizations and increased in titer against each reactive melanoma cell line during the immunizations. Reactivity of this serum with melanoma cell lines demonstrated a direct correlation with the expression of GD3 by the respective cell line. Anti-GD3 reactivity was evident in solid-phase radioimmunoassay against purified GD3 beginning with serum collected after 11 immunizations. By comparison with its binding to the control ganglioside panel, this serum demonstrated strong specificity for GD3 (titer=640) while having only marginal reactivity with GM3 (titer=40). Immune serum from this animal was also able specifically to block subsequent binding of a murine IgM anti-GD3 antibody (DMab7) to target GD3 in solid-phase radioimmunoassay. Together, these observations suggest that GD3, in the form of a purified molecule bound to a bacterial matrix or as part of the intact melanoma cell membrane, can be immunogenic in non-human primates, and is able to elicit an antibody response of appropriate specificity.Supported in part by grant CA32672 from the National Cancer Institute, Veterans Administration Program 821 and by the Yerkes Regional Primate Center, Atlanta, Georgia. The Yerkes Center is fully accredited by the American Association for Accreditation of Laboratory Animal Care 相似文献
88.
Gary Gibson Pamela Nielsen Victoria Mykytyn Ken Carlson John Blass 《Neurochemical research》1989,14(1):17-24
To further elucidate the molecular basis of the selective damage to various brain regions by thiamin deficiency, changes in enzymatic activities were compared to carbohydrate flux through various pathways from vulnerable (mammillary bodies and inferior colliculi) and nonvulnerable (cochlear nuclei) regions after 11 or 14 days of pyrithiamin-induced thiamin deficiency. After 11 days,large decreases (–43 to –59%) in transketolase (TK) occurred in all 3 regions; 2-ketoglutarate dehydrogenase (KGDHC) declined (–45%), but only in mammillary bodies; pyruvate dehydrogenase (PDHC) was unaffected. By day 14, TK remained reduced by 58%–66%; KGDHC was now reduced in all regions (–48 to –55%); PDHC was also reduced (–32%), but only in the mammillary bodies. Thus, the enzyme changes did not parallel the pathological vulnerability of these regions to thiamin deficiency.14CO2 production from14C-glucose labeled in various positions was utilized to assess metabolic flux. After 14 days, CO2 production in the vulnerable regions declined severely (–46 to 70%) and approximately twice as much as those in the cochlear nucleus. Also by day 14, the ratio of enzymatic activity to metabolic flux increased as much as 56% in the vulnerable regions, but decreased 18 to 30% in the cochlear nuclei. These differences reflect a greater decrease in flux than enzyme activities in the two vulnerable regions. Thus, selective cellular responses to thiamin deficiency can be demonstrated ex vivo, and these changes can be directly related to alterations in metabolic flux. Since they cannot be related to enzymatic alterations in the three regions, factors other than decreases in the activity of these TPP-dependent enzymes must underlie selective vulnerability in this model of thiamin deficiency.Abbreviations KGDHC
2-ketoglutarate dehydrogenase complex EC 1.2.4.2., EC 2.3.1.61, EC 1.6.4.3.
- PDHC
pyruvate dehydrogenase complex EC 1.2.4.2., EC 2.3.1.12, EC 1.6.4.3
- TK
transketolase (EC 2.2.1.1)
- TPP
thiamin pyrophosphate 相似文献
89.
A single cDNA encodes two isoforms of stathmin, a developmentally regulated neuron-enriched phosphoprotein 总被引:6,自引:0,他引:6
V Doye F Soubrier G Bauw M C Boutterin L Beretta J Koppel J Vandekerckhove A Sobel 《The Journal of biological chemistry》1989,264(21):12134-12137
Stathmin, a 19-kDa neuron-enriched soluble phosphoprotein, has been recently proposed as an ubiquitous intracellular relay for the diverse extracellular signals regulating cell proliferation, differentiation, and functions through various second messenger pathways (Sobel, A., Boutterin, M.C., Beretta, L., Chneiweiss, H., Doye, V., and peyro-Saint-Paul, H. (1989) J. Biol. Chem. 264, 3765-3772). Internal sequences of the protein from rat brain were determined after purification by two-dimensional polyacrylamide gel electrophoresis, electrotransfer onto Immobilon, and in situ proteolysis. Oligonucleotide mixtures based on these sequences were used to clone a cDNA for stathmin from a rat PC12 cell lambda gt 10 library. The deduced amino acid sequence reveals partial homologies with the coiled coil structural regions of several intracellular matrix phosphoproteins. Using this cDNA as a probe, we show that the expression of stathmin mRNA parallels that of the protein during brain ontogenesis, reaching a maximum at the neonatal stage. In vitro translation of the derived cRNA yielded all the known molecular forms of stathmin, namely its alpha and beta isoforms in their unphosphorylated and phosphorylated states. Thus, a single cDNA codes for both biologically relevant isoforms of the protein, indicating that they differ by co- or post-translational modifications. 相似文献
90.
Molecular and functional analysis of the ruv region of Escherichia coli K-12 reveals three genes involved in DNA repair and recombination 总被引:14,自引:0,他引:14
Gary J. Sharples Fiona E. Benson Graham T. Illing Robert G. Lloyd 《Molecular & general genetics : MGG》1990,221(2):219-226
Summary Recombinant plasmids carrying ruvA, ruvB, or both were constructed and used to investigate the genetic defects in a collection of UV-sensitive ruv mutants. The results revealed that efficient survival of UV-irradiated cells depends on both ruvA and ruvB, and on a third gene, ruvC, located upstream of the ruvAB operon. Southern blotting analysis was used to locate insertions in ruv and to examine putative deletion mutants. Two Tn10 insertions were located to the region encoding ruvA. Since these insertions caused a deficiency in the activities of both ruvA and ruvB, we concluded that they must exert a polar effect on ruvB. Two putative ruv deletion mutants were shown to be the result of deletion-inversion events mediated during imprecise excision of Tn10. The relevant inversion breakpoints in these mutants were located to ruvA and ruvC. 相似文献