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91.
Characterization of SKM1, a Saccharomyces cerevisiae gene encoding a novel Ste20/PAK-like protein kinase 总被引:1,自引:0,他引:1
Humberto Martín Alfonso Mendoza Jose M. Rodríguez-Pachón María Molina & César Nombela 《Molecular microbiology》1997,23(3):431-444
Ste20/PAK serine/threonine protein kinases have been suggested as playing essential roles in cell signalling and morphogenesis as potential targets of Cdc42 and Rac GTPases. We have isolated and characterized the Saccharomyces cerevisiae SKM1 gene, which codes for a novel member of this family of protein kinases. The amino acid sequence analysis of Skm1p revealed the presence of a PH domain and a putative p21-binding domain near its amino terminus, suggesting its involvement in cellular signalling or cytoskeletal functions. However, deletion of SKM1 produced no detectable phenotype under standard laboratory conditions. Moreover, disruption of each of the two other S. cerevisiae Ste20/PAK-like kinase-encoding genes, STE20 and CLA4 , in skm1 backgrounds, showed that Skm1p is not redundant with Ste20p or Cla4p. Interestingly, overexpression of SKM1 led to morphological alterations, indicating a possible role for this protein in morphogenetic control. Furthermore, overproduction of Skm1p lacking its N-terminus caused growth arrest. This effect was also seen when similarly truncated versions of Ste20p or Cla4p were overexpressed. We further observed that overproduction of this C-terminal fragment of Skm1p complements the mating defect of a ste20 mutant strain. These results suggest that the N-terminal domains of S. cerevisiae Ste20/PAK-like protein kinases share a negative regulatory function and play a role in substrate specificity. 相似文献
92.
93.
Isabel Haro Rosa M. Pinto Juan F. Gonzalez-Dankaart Jose A. Perez Francisca Reig Albert Bosch 《Microbiology and immunology》1995,39(7):485-490
Peptide VP1 (11-25) of the capsid of hepatitis A virus was synthesized by the Fmoc-polyamide solid phase method, and administered to mice in different forms: (1) free, (2) encapsulated in multilamellar liposomes, (3) coupled to keyhole limpet hemocyanin (KHL), and (4) incorporated into a tetrameric branched lysine core. The highest anti-VP1 peptide responses were generated by synthetic peptides entrapped into liposomes and coupled to KLH. No anti-HAV response was generated with the free peptide, while all the other forms induced both anti-HAV and HAV-neutralizing antibodies. Maximum neutralization indices were observed in ascites from mice treated with liposome-entrapped and KLH peptides. 相似文献
94.
Cecilia Ballaré Marcela Barrio Paula Portela Jose Mordoh 《Cancer immunology, immunotherapy : CII》1995,41(1):15-22
FC-2.15 is a murine IgM monoclonal antibody (mAb) that recognizes a cell-surface antigen (Ag2.15) expressed in most tumor-proliferating cells of human breast carcinomas and other neoplasias. In this study the cytotoxic ability of mAb FC-2.15, its cell-surface binding properties and endocytosis in Ag2.15-expressing (Ag2.15+) cells were investigated. A51Cr-release assay was used to test the FC-2.15-mediated cytotoxicity. When human serum was used as source of complement, FC-2.15 exerted a strong cytotoxic effect against human Ag2.15+ cells such as MCF-7 (breast cancer cell line), primary breast carcinoma cells, polymorphonuclear leukocytes and chronic myeloid leukemia cells. The mAb concentration range was 1–50 g/ml. Cytotoxicity was completely abolished when complement was inactivated. Only 3.8±2.9% of MCF-7 cells survived the treatment with FC-2.15 in the presence of human serum. A flow-cytometry assay was performed to study the Ag2.15 expression of the surviving cells and they were found to be Ag2.15–. FC-2.15 did not mediate antibody-dependent cell cytotoxicity when different effector cells were used. Scatchard analysis with125I-FC-2.15 on MCF-7 cells demonstrated an affinity constant of 6.9×107 M–1 and 2.8×106 antigenic sites/cell.125I-FC-2.15 was internalized to cytoplasmic vesicles reaching a maximum of 27% after 6 h incubation, followed by the release of labeled degradation products to the supernatant. FC-2.15 appears to exert its cytotoxic effect mainly in the presence of human complement, it reacts with intermediate affinity with a high-density surface antigen, and it is slowly internalized by Ag2.15+ cells. 相似文献
95.
Bernardo Celda Clelia Biamonti Maria Jose Arnau Roberto Tejero Gaetano T. Montelione 《Journal of biomolecular NMR》1995,5(2):161-172
Summary A large portion of the 13C resonance assignments for murine epidermal growth factor (mEGF) at pH 3.1 and 28°C has been determined at natural isotope abundance. Sequence-specific 13C assignments are reported for 100% of the assignable C, 96% of the C, 86% of the aromatic and 70% of the remaining peripheral aliphatic resonances of mEGF. A good correlation was observed between experimental and back-calculated C chemical shifts for regions of regular -sheet structure. These assignments also provide the basis for interpreting 1H–13C heteronuclear NOE (HNOE) values in mEGF at natural isotope abundance. Some of the backbone polypeptide segments with high internal mobility, indicated by these 1H–13C HNOE measurements, correlate with locations of residues involved in the putative mEGF-receptor binding site. Using four families of mEGF structures obtained over the last few years, we demonstrate that standard deviations between experimental and back-calculated C values can be used to monitor the refinement of this protein's structure, particularly for -sheet regions. Improved agreement between calculated and observed values of C is correlated with other measures of structure quality, including lowered values of residual constraint violations and more negative values of conformational energy. These results support the view that experimental conformation-dependent chemical shifts, C, can provide a reliable source of information for monitoring the process of protein structure refinement and are potentially useful restraints for driving the refinement.Abbreviations HSQC
heteronuclear single-quantum coherence spectroscopy
- PFG
pulsed-field gradient
- TOCSY
1H-1H total correlation spectroscopy
- EGF
epidermal growth factor
- mEGF
murine EGF
- hEGF
human EGF
- hTGF
human type- transforming growth factor
- DIPSI
spm-locking pulse sequence
- NOE
nuclear Overhauser effect
- HNOE
heteronuclear Overhauser effect 相似文献
96.
Characterization of a plasma membrane-associated phosphoinositide-specific phospholipase C from soybean 总被引:12,自引:1,他引:11
Jinrui Shi Robert A. Gonzales Madan K. Bhattacharyya 《The Plant journal : for cell and molecular biology》1995,8(3):381-390
Phosphoinositide-specific phospholipase C (PI-PLC) is a key signal transducing enzyme which generates the second messengers inositol trisphosphate and diacylglycerol in mammalian cells. A cDNA clone (PI-PLC1) encoding a phosphoinositide-specific phospholipase C was isolated from soybean by screening a cDNA expression library using an anti-(plasma membrane) serum. Genomic DNA gel blot analysis suggested that the corresponding gene is a member of a multigene family. The deduced amino acid sequence of the soybean PI-PLC1 isozyme contains the conserved X and Y regions, found in other PI-PLCs. It is closely related to mammalian δ-type PI-PLCs, Dictyostelium discoideum PI-PLC and yeast PI-PLC1 in terms of the arrangement of the conserved region. Unlike mammalian δ-type PI-PLCs and yeast PI-PLC1, the putative Ca2+ -binding site of the soybean PI-PLC1 is located in the region spanning the X and Y domains, and the N-terminal region is truncated. FLAG epitope-tagged PI-PLC1 fusion protein purified from transgenic tobacco plants showed phosphoinositide-specific phospholipase C activity. Heterologous expression of the soybean PI-PLC1 cDNA in a yeast PI-PLC1 deletion mutant complemented the lethality phenotype of haploid PI-PLC1 disruptants. Immunoblot analysis of the cell fractions prepared from transgenic tobacco plants over-expressing the FLAG epitope-tagged PI-PLC1 fusion protein indicated that the protein encoded by the PI-PLC1 cDNA was localized in the cytosol and plasma membrane. 相似文献
97.
Jose L. Garcia-Martinez Noah Ben-Shalom Lawrence Rappaport 《Plant Growth Regulation》1984,2(3):209-216
The effect of gibberellin A1 (GA1) on production of ethylene by cowpea (Vigna sinensis cv Blackeye pea no. 5) epicotyl explants and its relationship to epicotyl elongation was investigated. The explants were placed upright in water and incubated in sealed culture tubes or in large jars. GA, and IAA in ethanol solution were injected into the subapical tissues of the decapitated epicotyls. Cowpea epicotyl explants elongated after GA but not after IAA treatment, and they were very sensitive to exogenous ethylene. As little as 0.14 1/1 ethylene reduced significantly GA1-induced epicotyl elongation.Treatment with GA1 induced the production of ethylene which began 10 h after GA application, showed a peak at about 22 h and then declined. The yield of ethylene was proportional to the amount of GA, injected. The inhibition of epicotyl elongation in closed tubes was avoided by absorbing ethylene released with Hg(Cl04)2 , or by adding AVG to the incubation solution to inhibit ethylene production. Treatment with IAA elicited a rapid production of ethylene which ceased about 10 h after application. The effects of IAA and GA1 on ethylene production were additive.Abbreviations AVG
aminoethoxyvinylglycine 2-amino-4-(2-aminoethoxy)-trans-3butenoic acid
- ACC
1-aminocyclopropane-1-carboxylic acid
- GA
gibberellin
- IAA
indole-3-acetic acid 相似文献
98.
Ernesto Bonilla Enrique Salazar Jose Joaquin Villasmil Ruddy Villalobos Magaly Gonzalez Jose Omar Davila 《Neurochemical research》1984,9(11):1543-1548
Copper concentration was determined in samples from 38 areas of 7 normal human brains. The grey matter contained higher concentrations of copper than the white matter. Identical areas of the grey and white matter of the cerebral cortex showed significant differences between individuals. In the caudate nucleus the highest concentrations of copper were found in the tail followed by the body and the head, respectively. A negative linear regression between age and brain copper levels was demonstrated. 相似文献
99.
100.
Freeze fracturing electron microscopy of Escherichia coli K12 cells showed that the outer fracture face of the outer membrane is densily occupied with particles. On the inner fracture face of the outer membrane, pits are visible, which are probably complementary to the particles at opposite fracture face. This observation suggests that the particles are micelle-like. In some mutants which lack one or more major outer membrane proteins the density of particles is reduced. The loss of protein d appeared to a prerequisite for this phenomenon. However, mutants which lack all glucose and heptose-bound phosphate in their lipopolysaccharide also have a reduction in particle density whereas, the amount of protein d is normal. Moreover, loss of lipopolysaccharide by EDTA treatment also caused a reduction in the density of particles. From these results it is hypothesized that the particles consist of lipopolysaccharide aggregates stabilized by divalent cations and probably complexed with protein and/or phospholipid. 相似文献