Notch is a single-pass transmembrane receptor protein. Delta (member of the DSL protein family), a Notch ligand, is also single-pass transmembrane protein that can interact with Notch to form the Delta-Notch complex. It has been demonstrated that of the 36 Epidermal Growth Factor (EGF) repeats of Notch, 11th and 12th are sufficient to mediate interactions with Delta. Crystal structure of mammalian Notch1 extracellular ligand binding domain shows the presence of 11th and 12th EGF-like repeats. Here a portion of the Drosophila Delta protein, known to interact with Notch extracellular domain, has been modeled using homology modeling. The structure of the Delta-Notch complex was subsequently modeled by protein-docking method using GRAMM. Molecular dynamic simulations of the modeled structures were performed. The probable structures for Delta-Notch complex have been proposed based on interaction energy parameter and planarity studies. 相似文献
Defensins are antimicrobial peptides that are important in the innate immune defense of mammals. In contrast to mammalian α- and β-defensins, rhesus θ-defensin-1 (RTD-1) comprises only 18 amino acids stabilized by three disulfide bonds and an unusual backbone cyclic topology. In this work we report for the first time the recombinant expression of the fully folded θ-defensin RTD-1 using a bacterial expression system. This was accomplished using an intramolecular native chemical ligation in combination with a modified protein-splicing unit. RTD-1 was produced either in vitro or in vivo. In-cell production of RTD-1 was estimated to reach an intracellular concentration of ~4 μM. Recombinant RTD-1 was shown to be correctly folded as characterized by heteronuclear-NMR and by its ability to specifically inhibit lethal factor protease. The recombinant production of folded θ-defensins opens the possibility to produce peptide libraries based on this peptide scaffold that could be used to develop in-cell screening and directed evolution technologies. 相似文献
The broad range of applications of spatially‐offset Raman spectroscopy (SORS) were found to involve samples having only marginal differences in Raman cross‐sections between the surface and subsurface targets. We report the results of a feasibility study to evaluate the potential of the approach to identify the presence of a very low Raman‐active turbid sample placed inside a highly Raman‐active diffusely scattering matrix. Paraffin sandwiched tissue blocks prepared by embedding slices of chicken muscle tissue into solid paraffin blocks were employed as representative samples for the study. It was found that in contrast to the several millimetres of probing depth reported in the earlier applications, the Raman signatures of tissue were best recovered when it was located beneath the surface of the paraffin block at a depth of around a millimetre, beyond which the quality of recovery was increasingly poorer. However, the probing depth could be further increased by increasing the thickness of the embedded tissue sections. The results clearly suggest that though the probing depth achievable under the current condition is less than that found in previous applications, nevertheless it is sufficient for various other applications that would not require probing as deep as was required earlier.
Protein kinase C (PKC) is an important constituent of the signaling pathways involved in apoptosis. We report here that like staurosporine, withaferin A is a potent inhibitor of PKC. In Leishmania donovani, the inhibition of PKC by withaferin A causes depolarization of DeltaPsim and generates ROS inside cells. Loss of DeltaPsim leads to the release of cytochrome c into the cytosol and subsequently activates caspase-like proteases and oligonucleosomal DNA cleavage. Moreover, in treated cells, oxidative DNA lesions facilitate the stabilization of topoisomerase I-mediated cleavable complexes, which also contribute to DNA fragmentation. However, withaferin A and staurosporine cannot induce cleavable complex formation in vitro with recombinant topoisomerase I nor with nuclear extracts from control cells. Taken together, our results indicate that inhibition of PKC by withaferin A is a central event for the induction of apoptosis and that the stabilization of topoisomerase I-DNA complex is necessary to amplify apoptotic process. 相似文献
BACKGROUND: The presently available cell motility-analyzers measure primarily the "horizontal" velocity and there is no instrument available for "vertical" velocity measurement. This development was based on the turbidimetric method of sperm motility analysis. METHODS: Sperm was layered at the bottom of the cuvette containing buffer solution and exposed to the spectrophotometric light path at different heights to track the vertically moving sperms. The vertical movement was materialized with the development of an electromechanical up-down movement devise for the cuvette accomplished with the help of a cuvette holder-stepper motor-computer assembly. The entire system was controlled by the necessary motion control, data acquisition, and data processing software developed for cuvette movement and data analysis. RESULTS: Using goat sperm as the model a unique computer-based spectrophotometric system has been developed for the first time to determine the average "vertical" velocity of motile cells. CONCLUSIONS: Undertaking upward movement against gravity is much tougher as compared with horizontal movement. Consequently average vertical velocity is expected to be a much better identifying parameter for assessing semen and other motile cell quality. The novel instrumental system developed by us has thus the potential for immense application in human infertility clinics, animal-breeding centres, centres for conservation of endangered species, and also for research work on vertical velocity of spermatozoa and other motile cells, such as bacteria, protozoa, etc. 相似文献
We explored the effect of copper availability on the synthesis and trafficking of peptidylglycine alpha-amidating monooxygenase (PAM), an essential cuproenzyme whose catalytic domains function in the lumen of peptide-containing secretory granules. Corticotrope tumor cell lines expressing integral membrane and soluble forms of PAM were depleted of copper using bathocuproinedisulfonic acid or loaded with copper by incubation with CuCl(2). Depleting cellular copper stimulates basal secretion of soluble enzyme produced by endoproteolytic cleavage of PAM in secretory granules and transit of membrane PAM though the endocytic pathway and back into secretory granules. Unlike many cuproenzymes, lack of copper does not lead to instability of PAM. Copper loading decreases cleavage of PAM in secretory granules, secretion of soluble enzyme, and the return of internalized PAM to secretory granules. The trafficking and stability of the soluble, luminal domain of PAM and truncated membrane PAM lacking a cytosolic domain are not affected by copper availability. Taken together, our data demonstrate a role for copper-sensitive cytosolic machinery in directing endocytosed membrane PAM back to secretory granules or to a degradative pathway. The response of PAM to lack of copper suggests that it facilitates copper homeostasis. 相似文献
All eukaryotic topoisomerase I enzymes are monomeric enzymes, whereas the kinetoplastid family (Trypanosoma and Leishmania) possess an unusual bisubunit topoisomerase I. To determine what happens to the enzyme architecture and catalytic property if the two subunits are fused, and to explore the functional relationship between the two subunits, we describe here in vitro gene fusion of Leishmania bisubunit topoisomerase I into a single ORF encoding a new monomeric topoisomerase I (LdTOPIL-fus-S). It was found that LdTOPIL-fus-S is active. Gene fusion leads to a significant modulation of in vitro topoisomerase I activity compared to the wild-type heterodimeric enzyme (LdTOPILS). Interestingly, an N-terminal truncation mutant (1-210 amino acids) of the small subunit, when fused to the intact large subunit [LdTOPIL-fus-Delta(1-210)S], showed reduced topoisomerase I activity and camptothecin sensitivity in comparison to LdTOPIL-fus-S. Investigation of the reduction in enzyme activity indicated that the nonconserved 1-210 residues of LdTOPIS probably act as a 'pseudolinker' domain between the core and catalytic domain of the fused Leishmania enzyme, whereas mutational analysis of conserved His453 in the core DNA-binding domain (LdTOPIL) strongly suggested that its role is to stabilize the enzyme-DNA transition state through hydrogen bonding to one of the nonbridging oxygens. Taken together, our findings provide an insight into the details of the unusual structure of bisubunit topoisomerase I of Leishmania donovani. 相似文献
Multidrug resistance (MDR) is still a major threat to successful clinical application of cancer chemotherapy. Copper plays
an important role in biological systems, and copper is also involved in carcinogenesis. In the present investigation, we addressed
the question whether metal copper might be involved in drug resistance of murine and human tumors. By means of atomic absorption
spectroscopy, we determined serum copper concentrations. We found that the blood serum of tumor-bearing mice contained higher
amounts of copper than healthy mice with tumors. Secondly, mice bearing doxorubicin-resistant Ehrlich ascites carcinoma- or
cyclophosphamide-resistant Lewis lung carcinoma contained more copper in their serum than mice bearing the corresponding drug-sensitive
parental tumors. Furthermore, the analysis of patients with breast cancer, colon carcinoma or lung cancer showed that the
serum copper contents were higher in patients not responding to chemotherapy when compared to patients whose tumors responded
to treatment. The copper levels in serum of healthy volunteers were lower than in cancer patients irrespective of their response
to chemotherapy. Our results imply that the level of serum copper may be considered as a biomarker for treatment response. 相似文献
Salinity poses a serious threat to yield performance of cultivated rice in South Asian countries. To understand the mechanism
of salt-tolerance of the wild halophytic rice, Porteresia coarctata in contrast to the salt-sensitive domesticated rice Oryza sativa, we have compared P. coarctata with the domesticated O. sativa rice varieties under salinity stress with respect to several physiological parameters and changes in leaf protein expression.
P. coarctata showed a better growth performance and biomass under salinity stress. Relative water content was conserved in Porteresia during stress and sodium ion accumulation in leaves was comparatively lesser. Scanning electron microscopy revealed presence
of two types of salt hairs on two leaf surfaces, each showing a different behaviour under stress. High salt stress for prolonged
period also revealed accumulation of extruded NaCl crystals on leaf surface. Changes induced in leaf proteins were studied
by two-dimensional gel electrophoresis and subsequent quantitative image analysis. Out of more than 700 protein spots reproducibly
detected and analyzed, 60% spots showed significant changes under salinity. Many proteins showed steady patterns of up- or
downregulation in response to salinity stress. Twenty protein spots were analyzed by MALDI-TOF, leading to identification
of 16 proteins involved in osmolyte synthesis, photosystem functioning, RubisCO activation, cell wall synthesis and chaperone
functions. We hypothesize that some of these proteins confer a physiological advantage on Porteresia under salinity, and suggest a pattern of salt tolerance strategies operative in salt-marsh grasses. In addition, such proteins
may turn out to be potential targets for recombinant cloning and introgression in salt-sensitive plants.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献