Identification of a ubiquitin-binding structure in the S-locus F-box protein controlling S-RNase-based self-incompatibility

In flowering plants,self-incompatibility(SI) serves as an important intraspecific reproductive barrier to promote outbreeding.In species from the Solanaceae,Plantaginaceae and Rosaceae,S-RNase and SLF(S-locus F-box) proteins have been shown to control the female and male specificity of SI,respectively.However,little is known about structure features of the SLF protein apart from its conserved F-box domain.Here we show that the SLF C-terminal region possesses a novel ubiquitin-binding domain(UBD) structure conserved among the SLF protein family.By using an ex vivo system of Nicotiana benthamiana,we found that the UBD mediates the SLF protein turnover by the ubiquitin—proteasome pathway.Furthermore,we detected that the SLF protein was directly involved in S-RNase degradation.Taken together,our results provide a novel insight into the SLF structure and highlight a potential role of SLF protein stability and degradation in S-RNase-based self-incompatibility.     

Comparison of Petunia inflata S-Locus F-box protein (Pi SLF) with Pi SLF like proteins reveals its unique function in S-RNase based self-incompatibility

Petunia inflata possesses S-RNase-based self-incompatibility (SI), which prevents inbreeding and promotes outcrossing. Two polymorphic genes at the S-locus, S-RNase and P. inflata S-locus F-box (Pi SLF), determine the pistil and pollen specificity, respectively. To understand how the interactions between Pi SLF and S-RNase result in SI responses, we identified four Pi SLF-like (Pi SLFL) genes and used them, along with two previously identified Pi SLFLs, for comparative studies with Pi SLF(2). We examined the in vivo functions of three of these Pi SLFLs and found that none functions in SI. These three Pi SLFLs and two other Pi SLFs either failed to interact with S(3)-RNase (a non-self S-RNase for all of them) or interacted much more weakly than did Pi SLF(2) in vitro. We divided Pi SLF(2) into FD1 (for Functional Domain1), FD2, and FD3, each containing one of the Pi SLF-specific regions, and used truncated Pi SLF(2), chimeric proteins between Pi SLF(2) and one of the Pi SLFLs that did not interact with S(3)-RNase, and chimeric proteins between Pi SLF(1) and Pi SLF(2) to address the biochemical roles of these three domains. The results suggest that FD2, conserved among three allelic variants of Pi SLF, plays a major role in the strong interaction with S-RNase; additionally, FD1 and FD3 (each containing one of the two variable regions of Pi SLF) together negatively modulate this interaction, with a greater effect on interactions with self S-RNase than with non-self S-RNases. A model for how an allelic product of Pi SLF determines the fate of its self and non-self S-RNases in the pollen tube is presented.     

Evaluation of the Sterifil Lysis-Filtration Blood Culture System

This paper describes the comparison of the Sterifil lysis-filtration (SLF) blood culture procedure with a standard Trypticase soy broth (TSB) technique. The lysing solutions employed in the SLF system, Triton X-100 (alkyl phenoxy polyethoxy ethanol) and sodium carbonate, were deleterious to most bacteria commonly encountered in bacteremia except staphylococci and enterococci. Candida was not adversely affected. There was a positive correlation between the tolerance of the microbial isolants to the lysing solutions and their recovery by the SLF technique. A total of 3,554 cultures were run in parallel and 398 isolants were obtained. Of 201 gram-positive isolants, 135 were recovered by both techniques, 43 were detected by the TSB technique only, and 23 were recovered only with the SLF method. In sharp contrast, of 168 gram-negative isolants, 28 were recovered in common, 130 were isolated only by TSB, and 10 were recovered only with the SLF method. The SLF method detected all cases of candidemia detected by the TSB method plus an additional 12 for a total of 29 cases. The SLF method, as currently described, is generally too toxic to bacteria for routine use in a clinical laboratory.     

Effect of seaweed liquid fertilizer on yield and quality of Capsicum annum L.

In the present study, intensive investigation was made on the effect of seaweed liquid fertilizer (SLF) of Codium decorticatum on the seed germination yield biochemical and pigment characteristic of Capsium annum under laboratory conditions and in pots. Different concentrations such as 10%, 20%, 30%, 40% and 50% of SLF were prepared using distilled water. The seeds were soaked in 10 h for each SLF concentration then placed in separate Petri plates. Similarly, water soaked seeds were used as controls. Application of a lower concentration (20%) of SLF Showed maximum seed germination, fresh weight, dry weight, root and shoot length, number of branches, leaf area, number of pods and content of total chlorophyll, chl a, and chl b, protein, carbohydrate and lipids were observed. Therefore, the results of the present study suggested that the SLF of C. decortianum could serve as an alternative bio-fertilizer as is eco-friendly, cost-effective, deliver substantial economic and environmental benefits to farmer.     

Expression of the mRNA for the ligand of c-kit in mouse Sertoli cells.

The expression of the mRNA for SLF (the c-kit ligand), a product of the "steel" locus, has been investigated in postnatal mouse testis and homogeneous populations of testicular cells. The message was found expressed in postnatal mouse testis but not in germ cells. Studies on primary mouse Sertoli cell cultures from 18 day old mice show that Sertoli cells are the site of SLF mRNA expression in the seminiferous tubules. Treatment of Sertoli cell cultures with cAMP analogs led to a significant increase in the SLF mRNA levels.     

Structural studies of tropomyosin by cryoelectron microscopy and x-ray diffraction.

A comparison has been made between cryoelectron microscope images and the x-ray structure of one projection of the Bailey tropomyosin crystal. The computed transforms of the electron micrographs extend to a resolution of approximately 18 A compared with the reflections from x-ray crystallography which extend to 15 A. After correction of the images for lattice distortions and the contrast transfer function, the structure factors were constrained to the plane group (pmg) symmetry of this projection. Amplitude and phase data for five images were compared with the corresponding view from the three-dimensional x-ray diffraction data (Phillips, G.N., Jr., J.P. Fillers, and C. Cohen. 1986. J. Mol. Biol. 192: 111-131). The average R factor between the electron microscopy and x-ray amplitudes was 15%, with an amplitude-weighted mean phase difference of 4.8 degrees. The density maps derived from cryoelectron microscopy contain structural features similar to those from x-ray diffraction: these include the width and run of the filaments and their woven appearance at the crossover regions. Preliminary images obtained from frozen-hydrated tropomyosin/troponin cocrystals suggest that this approach may provide structural details not readily obtainable from x-ray diffraction studies.     

Pollen S–locus F–box proteins of Petunia involved in S–RNase‐based self‐incompatibility are themselves subject to ubiquitin‐mediated degradation

Many flowering plants show self‐incompatibility, an intra‐specific reproductive barrier by which pistils reject self‐pollen to prevent inbreeding and accept non‐self pollen to promote out‐crossing. In Petunia, the polymorphic S–locus determines self/non‐self recognition. The locus contains a gene encoding an S–RNase, which controls pistil specificity, and multiple S‐locus F‐box (SLF) genes that collectively control pollen specificity. Each SLF is a component of an SCF (Skp1/Cullin/F‐box) complex that is responsible for mediating degradation of non‐self S‐RNase(s), with which the SLF interacts, via the ubiquitin–26S proteasome pathway. A complete set of SLFs is required to detoxify all non‐self S‐RNases to allow cross‐compatible pollination. Here, we show that SLF1 of Petunia inflata is itself subject to degradation via the ubiquitin–26S proteasome pathway, and identify an 18 amino acid sequence in the C‐terminal region of S₂‐SLF1 (SLF1 of S₂ haplotype) that contains a degradation motif. Seven of the 18 amino acids are conserved among all 17 SLF proteins of S₂ haplotype and S₃ haplotype involved in pollen specificity, suggesting that all SLF proteins are probably subject to similar degradation. Deleting the 18 amino acid sequence from S₂‐SLF1 stabilized the protein but abolished its function in self‐incompatibility, suggesting that dynamic cycling of SLF proteins is an integral part of their function in self‐incompatibility.     

Metabonomic study of Chinese medicine Shuanglong formula as an effective treatment for myocardial infarction in rats

A UPLC/TOF-MS-based metabonomic study was conducted to assess the holistic efficacy of Traditional Chinese Medicine Shuanglong Formula (SLF) for myocardial infarction in rats. Thirty male Sprague-Dawley rats were randomly divided into five groups after surgery. The Panax ginseng group, Salvia miltiorrhiza group, and SLF group were treated with water extractions of Panax ginseng (PG), Salvia miltiorrhiza (SM), and SLF (the ratio of SM to PG was 3:7) at a dose of 5 g/kg·w·d for 21 consecutive days, respectively; the model group and sham surgery group were both treated with 0.9% saline solution. Urinary samples for metabonomic study, serum samples for biochemical measurement, and heart samples for histopathology were collected. As a result, metabonomics-based findings such as the PCA and PLS-DA plotting of metabolic state and analysis of potential biomarkers in urine correlated well to the assessment of serum biochemistry and histopathological assay, confirming that SLF exerted synergistic therapeutic efficacies to exhibit better effect on MI compared to PG or SM. The shifts in urinary TCA cycle as well as pentose phosphate pathway suggested that SLF may diminish cardiac injury of MI with its potential pharmacological effect in the regulation of myocardial energy metabolism.     

Aging effects of motor prediction on protective balance and startle responses to sudden drop perturbations

This pilot study investigated the effect of age on the ability of motor prediction during self-triggered drop perturbations (SLF) to modulate startle-like first trial response (FTR) magnitude during externally-triggered (EXT) drop perturbations. Ten healthy older (71.4 ± 1.44 years) and younger adults (26.2 ± 1.63 years) stood atop a moveable platform and received blocks of twelve consecutive EXT and SLF drop perturbations. Following the last SLF trial, participants received an additional EXT trial spaced 20 min apart to assess retention (EXT RTN) of any modulation effects. Electromyographic (EMG) activity was recorded bilaterally over the sternocleidomastoid (SCM), vastus lateralis (VL), biceps femoris (BF), medial gastrocnemius (MG), and tibialis anterior (TA). Whole-body kinematics and kinetic data were recorded. Stability in the antero-posterior direction was quantified using the margin of stability (MoS). Compared with EXT trials, both groups reduced SCM peak amplitude responses during SLF and EXT RTN trials. VL/BF and TA/MG coactivation were reduced during SLF FTR compared to EXT FTR (p < 0.05) with reduced peak vertical ground reaction forces (vGRF) in both younger and older adults (p < 0.05). Older adults increased their MoS during SLF FTR compared to EXT FTR (p < 0.05). Both groups performed more eccentric work during SLF trials compared to EXT (p < 0.05). These findings indicate that abnormal startle effects with aging may interfere with balance recovery and increase risk of injury with external balance perturbations. Motor prediction may be used to acutely mitigate abnormal startle/postural responses with aging.     

Purification and characterization of the F1 portion of the ATP synthase (F1Fo) of Streptomyces lividans

The F1 complex of the ATP synthase of Streptomyces lividans was isolated and purified. The procedure involved the solubilization of F1 from membranes with buffer of low ionic strength in the presence of EDTA, ion-exchange chromatography and gel filtration. The purified F1 complex from S. lividans (SLF1) consists of five subunits alpha, beta, gamma, delta and epsilon with molecular masses of 58,000, 50,000, 36,000, 28,000 and 13,000, respectively and exhibits immunological cross-reactivity with the F1 portion purified from Escherichia coli (ECF1). The enzymatic properties of SLF1 were determined by the use of microtiter-plate-based assay and compared with data obtained for ECF1. ATPase activity of SLF1 (specific activity: 20-30 U/mg) was only observed in the presence of high concentrations of Ca2+ (10mM). Stimulation of the ATPase activity by Mg2+ was not detectable; quite to the contrary, Mg2+ inhibited the Ca(2+)-stimulated activity of SLF1. SLF1 was re-bound to F1-stripped membranes of S. lividans, but not to F1-stripped membrane vesicles of E. coli. In contrast, ECF1 could be cross-reconstituted with F1-stripped membranes of S. lividans; however, a structural but not a functional reconstitution of the hybrid F1Fo complex was observed.     

Effect of a pseudovolume image in multifocus radiography

The effect of a pseudovolume image was theoretically substantiated on the basis of calculations. This was experimentally studied using with microfocus x-ray diagnostic apparatuses. Digital microfocus and standard x-ray study were compared using an experimental material. Microfocus x-ray study is a highly informative technique that shows a greater possibility to provide volume images of an object than does standard x-ray study.     

Epitope mapping and functional studies with three monoclonal antibodies to the C-KIT receptor tyrosine kinase,YB5.B8, 17F11, and SR-1

Three monoclonal antibodies (MAbs) to the human c-kit receptor tyrosine kinase (P145^c-kit), derived in independent laboratories, have been extensively used in studies of c-kit expression and the role of its ligand, steel factor (SLF), in hemopoiesis and mast cell differentiation and function. In this study, the relationship between the epitopes they identify, and their effects on SLF binding, receptor internalization, and signal transduction are compared. Epitope mapping studies carried out on the high P145^c-kit-expressing cell line HEL-DR showed that SR-1 identifies an epitope independent of those bound by YB5.B8 and 17F11, while the latter two antibodies bound to distinct but interacting epitopes. SR-1 potently blocked the binding of SLF to P145^c-kit on these cells and also on cells of the factor-dependent line MO7e. In contrast, YB5.B8 and 17F11 had minimal effects on ligand binding. Conversely, SLF partially blocked the binding of SR-1 and YB5.B8 to cells, while binding of 17F11 was actually enhanced by SLF on some target cells. Preincubation of HEL-DR and MO7e cells with MAbs prior to exposure to SLF revealed that 17F11 itself brought about partial down-regulation of P145^c-kit and did not inhibit SLF-mediated down-regulation. SR-1 caused minimal down-regulation and inhibited SLF-mediated receptor internalization. YB5.B8 had minimal effects on either cell line in this assay. To determine whether the antibodies had any agonist activity, they were compared with SLF for their ability to bring about receptor phosphorylation in intact MO7e cells. All three antibodies induced detectable tyrosine phosphorylation with 17F11 being the most effective, while YB5.B8 was the least effective. Finally, the ability of the antibodies to influence the proliferation of the MO7e cells was examined. As expected, SR-1 potently inhibited the proliferative response to SLF, while 17F11 weakly inhibited and YB5.B8 had negligible effect. In the absence of SLF both 17F11 and YB5.B8 displayed very weak but reproducible agonist activity. © 1994 Wiley-Liss, Inc.     

The Steel factor.

Steel factor (SLF) is a recently identified growth factor which is the gene product of the murine Steel locus and a ligand for the c-kit tyrosine kinase receptor, the product of the dominant white spotting locus (W). Defects at these genetic loci result in aberrant melanocyte, germ cell, and hematopoietic development. Both the receptor (c-kit) and the ligand (SLF) have been shown to undergo tissue-specific mRNA splicing to produce distinct isoforms which have unique biological functions. As predicted by the phenotype of these mutations, SLF influences the growth and differentiation of melanocytes, primordial germ cells, and a broad spectrum of cell types in the hematopoietic progenitor and stem cell hierarchy. SLF has also been shown to have effects on hematopoietic lineages not predicted by defects seen in the Steel mouse.     

Antifibrotic properties of caveolin-1 scaffolding domain in vitro and in vivo

Lung fibrosis involves the overexpression of ECM proteins, primarily collagen, by alpha-smooth muscle actin (ASMA)-positive cells. Caveolin-1 is a master regulator of collagen expression by cultured lung fibroblasts and of lung fibrosis in vivo. A peptide equivalent to the caveolin-1 scaffolding domain (CSD peptide) inhibits collagen and tenascin-C expression by normal lung fibroblasts (NLF) and fibroblasts from the fibrotic lungs of scleroderma patients (SLF). CSD peptide inhibits ASMA expression in SLF but not NLF. Similar inhibition of collagen, tenascin-C, and ASMA expression was also observed when caveolin-1 expression was upregulated using adenovirus. These observations suggest that the low caveolin-1 levels in SLF cause their overexpression of collagen, tenascin-C, and ASMA. In mechanistic studies, MEK, ERK, JNK, and Akt were hyperactivated in SLF, and CSD peptide inhibited their activation and altered their subcellular localization. These studies and experiments using kinase inhibitors suggest many differences between NLF and SLF in signaling cascades. To validate these data, we determined that the alterations in signaling molecule activation observed in SLF also occur in fibrotic lung tissue from scleroderma patients and in mice with bleomycin-induced lung fibrosis. Finally, we demonstrated that systemic administration of CSD peptide to bleomycin-treated mice blocks epithelial cell apoptosis, inflammatory cell infiltration, and changes in tissue morphology as well as signaling molecule activation and collagen, tenascin-C, and ASMA expression associated with lung fibrosis. CSD peptide may be a prototype for novel treatments for human lung fibrosis that act, in part, by inhibiting the expression of ASMA and ECM proteins.     

Calibration of x-ray densitometry for the measurement of coral skeletal density

A new method is proposed for the measurement of coral skeletal density by x-radiography. X-radiographs were made of sections cut from skeletons of massive corals of the genus Porites. Included on the x-ray film with each specimen were an aluminium step-wedge, a set of aragonite standards and several aluminium bars, all of measured thickness and density. The images on the developed x-ray film were scanned with a microdensitometer. Semilogarithmic plots of microdensitometer output voltage vs. thickness of the aluminium and aragonite standards provided characteristic curves, with initial linear slopes which were defined as relative linear absorption coefficients. These coefficients varied with the x-ray exposure and microdensitometer measurement conditions; however, they were consistent within one set of conditions. The relative linear absorption coefficients and densities of the standards, together with data for thickness of standard vs. film exposure, can be used to determine the density of coral specimens at points along microdensitometer traverses of their x-ray images. The bars of aluminium can be used to correct for the non-uniform irradiation of the x-ray film which is characteristic of x-ray machines.Contribution No. 299 from the Australian Institute of Marine Science     

Steel factor controls the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture

Mouse epidermal melanoblasts and melanocytes preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanocyte-proliferation medium (MDMD) and melanoblast-proliferation medium (MDMDF) supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF). Pure cultured primary melanoblasts and melanocytes were then further cultured with MDMD/MDMDF supplemented with steel factor (SLF) (keratinocyte depletion). SLF increased the number of melanoblasts and melanocytes as well as the proportion of differentiated melanocytes in the absence of keratinocytes. Flow cytometric analysis showed that melanoblasts and melanocytes in the S and G2/M phases of the cell cycle were increased by treatment with SLF. Moreover, an anti-SLF antibody added to MDMD/MDMDF from the initiation of the primary culture (in the presence of keratinocytes) inhibited the proliferation of melanoblasts and melanocytes as well as the differentiation of melanocytes. These results suggest that SLF is one of the keratinocyte-derived factors involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture in cooperation with cAMP elevator and bFGF.     

Proteolysis of the matricellular protein hevin by matrix metalloproteinase-3 produces a SPARC-like fragment (SLF) associated with neovasculature in a murine glioma model

The matricellular SPARC-family member hevin (Sparc-like 1/SPARCL-1/SC1/Mast9) contributes to neural development and alters tumor progression in a range of mammalian models. Based on sequence similarity, we hypothesized that proteolytic digestion of hevin would result in SPARC-like fragments (SLF) that affect the activity and/or location of these proteins. Incubation of hevin with matrix metalloproteinase-3 (MMP-3), a protease known to cleave SPARC, produced a limited number of peptides. Sequencing revealed the major proteolytic products to be SPARC-like in primary structure. In gliomas implanted into murine brain, a SLF was associated with SPARC in the neovasculature but not with hevin, the latter prominent in the astrocytes encompassed by infiltrating tumor. In this model of invasive glioma that involves MMP-3 activity, host-derived SLF was not observed in the extracellular matrix adjacent to tumor cells. In contrast, it occurred with its homolog SPARC in the angiogenic response to the tumor. We conclude that MMP-3-derived SLF is a marker of neovessels in glioma, where it could influence the activity of SPARC.     

Isolation and characterization of Kit-independent melanocyte precursors induced in the skin of Steel factor transgenic mice

Steel factor (SLF, also called KIT-ligand, mast cell growth factor, or stem cell factor) acting through the tyrosine kinase receptor KIT is thought to be indispensable for the early phase of melanocyte development both in vivo and in vitro . In the present study, Kit-independent precursor cells were generated in mice expressing exogenous SLF in their skin keratinocytes and were detected as pigmented spots after administration of Kit function-blocking antibody. We successfully purified these precursor or stem cells as Kit⁺CD45⁻ cells by flow cytometry. The purified cells showed normal but delayed differentiation into mature melanocytes, indicating the immature nature of Kit-independent precursors. The Kit-independent interfollicular population generated in SLF transgenic mice was suggested to be the counterpart of the follicular melanocyte stem cell based on the Kit-independent nature for their survival.