Insulin suppresses collagenase stimulatory effect of stratifin in dermal fibroblasts

A delicate balance between synthesis and degradation of extracellular matrix (ECM) by matrix metalloproteinases (MMPs) is an essential feature of tissue remodeling. We have recently demonstrated that keratinocyte releasable stratifin, also known as 14-3-3 sigma protein, plays a critical role in modulating collagenase (MMP-1) mRNA expression in human dermal fibroblasts. In this study, we further characterized the collagenase stimulatory effect of stratifin in dermal fibroblasts and evaluated its effect in the presence and absence of insulin. Our data indicate that stratifin increases the expression of collagenase mRNA more than 20-fold in dermal fibroblasts, grown in either Dulbecco's modified Eagle's medium (DMEM) plus 2% or 10% fetal bovine serum (FBS). Collagenase stimulatory effect of stratifin was completely blocked, when fibroblasts were cultured in test medium consisting of 50% keratinocyte serum-free medium (KSFM) and 50% DMEM. The collagenase suppressive effect of test medium was directly proportional to the volume of KSFM used. As this medium contained insulin, we then evaluated the collagenase stimulatory effect of stratifin in dermal fibroblasts in the presence and absence of insulin. The results revealed that stratifin significantly increased the expression of collagenase mRNA/18S (*p < 0.05, n = 3) ratio, while insulin significantly decreased the expression of collagenase mRNA/18S (*p < 0.05, n = 3) ratio. The insulin inhibitory effect on collagenase mRNA expression was time and dose dependent. The maximal inhibitory effect of insulin was seen at 36 h post treatment. In conclusion, stratifin stimulates the expression of collagenase mRNA expression in dermal fibroblasts and this effect is suppressed by insulin treatment.     

Species-specific variation in the importance of the spectral quality gradient in canopies as a signal for photosynthetic resource partitioning

BACKGROUND AND AIMS: Plants adjust the distribution of photosynthetic capacity and chlorophyll to canopy density. The importance of the gradient in the red : far-red ratio (R : FR) relative to the irradiance gradient was studied for its perception with respect to this partitioning of photosynthetic resources. Whether the relative importance of these two signals varied between six species of different growth habit (Phaseolus vulgaris, Lysimachia vulgaris, Hedera helix, Ficus benjamina, Carex acutiformis and Brachypodium pinnatum) was investigated further. METHODS: Single leaves of plants were shaded in daylight by a spectrally neutral filter or a leaf. In another experiment, leaves were treated with supplemental FR. In most cases, treatment effects were evaluated after 2 weeks. KEY RESULTS: Nitrogen and photosynthetic capacity (Amax) per leaf area, parameters pertaining to between-leaf resource partitioning, were strongly reduced in neutral shade but not additionally by spectral leaf shade. Supplemental FR reduced these parameters also, except in Carex. Acceleration of induction of senescence was observed in spectral leaf shade in primary bean leaves. Amax per unit chlorophyll, a parameter pertaining to within-leaf resource partitioning, was reduced in neutral shade, but not in spectral leaf shade or supplemental FR. CONCLUSIONS: Signalling mechanisms associated with perception of the R : FR gradient in canopies were less important than those associated with the irradiance gradient for between-leaf and within-leaf partitioning of photosynthetic resources. The relative importance of the signals differed between species because Carex was the only species for which no indications were found for an involvement of the spectral gradient in perception of canopy density.     

Dephosphorylation of cofilin is regulated through Ras and requires the combined activities of the Ras-effectors MEK and PI3K

Remodeling of the actin cytoskeleton is crucial for a multitude of cellular functions including cell movement, intracellular transport as well as signal transduction and gene expression processes. Cofilin has been identified as a key mediator of actin reorganization. Its activity is regulated via reversible phosphorylation of ser-3. In a variety of cell types stimulation through particular surface receptors fastly induces the dephosphorylation/activation of cofilin. Yet, the signal transduction cascades linking receptor stimulation with cofilin activation have not been identified so far. Here we show that the GTPase Ras acts as a central regulator of the cofilin dephosphorylation pathway. Thus, stimulation of Ras through platelet-derived growth factor (PDGF) or transient expression of activated Ras-proteins induces the dephosphorylation of cofilin. Importantly, the cooperation of two Ras-initiated signaling pathways is required to induce cofilin dephosphorylation: a Ras-Raf-MAPkinase/Erk-kinase (MEK)- and a Ras-phosphatidylinositol-3-kinase (PI3K)-effector cascade.     

Peroxisomal localization of sulfite oxidase separates it from chloroplast-based sulfur assimilation

Recently, we isolated the sulfite oxidase (SO) gene from Arabidopsis thaliana and characterized the purified SO protein. The purpose of the present study was to determine the subcellular localization of this novel plant enzyme. Immunogold electron-microscopic analysis showed the gold labels nearly exclusively in the peroxisomes. To verify this finding, green fluorescent protein was fused to full-length plant SO including the putative peroxisomal targeting signal 1 (PTS1) 'SNL' and expressed in tobacco leaves. Our results showed a punctate fluorescence pattern resembling that of peroxisomes. Co-labelling with MitoTracker-Red excluded that the observed fluorescence was due to mitochondrial sorting. By investigation of deleted or mutated PTS1, no functional peroxisomal targeting signal 2 (PTS2) could be detected in plant SO. This conclusion is supported by expression studies in Pichia pastoris mutants with defined defects either in PTS1- or PTS2-mediated peroxisomal import.     

The patient and observer scar assessment scale: a reliable and feasible tool for scar evaluation

At present, various scar assessment scales are available, but not one has been shown to be reliable, consistent, feasible, and valid at the same time. Furthermore, the existing scar assessment scales appear to attach little weight to the opinion of the patient. The newly developed Patient and Observer Scar Assessment Scale consists of two numeric scales: the Patient Scar Assessment Scale (patient scale) and the Observer Scar Assessment Scale (observer scale). The patient and observer scales have to be completed by the patient and the observer, respectively. The patient scale's consistency and the observer scale's consistency, reliability, and feasibility were tested. For the Vancouver Scar Scale, which is the most frequently used scar assessment scale at present, the same statistical measurements were examined and the results of the observer scale and the Vancouver scale were compared. The concurrent validity of the observer scale was tested with a correlation to the Vancouver scale. Furthermore, the authors examined which specific characteristics significantly influence the general opinion of the patient and the observers on the scar areas. Four independent observers have each used the observer scale and the Vancouver scale to assess 49 burn scar areas of 3 x 3 cm belonging to 20 different patients. Subsequently, the patients completed the patient scale for their scar areas. The (internal) consistency of both the patient and the observer scales was acceptable (Cronbach's alpha, 0.76 and 0.69, respectively), whereas the consistency of the Vancouver scale appeared not to be acceptable (alpha, 0.49). The reliability of the observer scale completed by a single observer was acceptable (r = 0.73). The reliability of the Vancouver scale completed by a single observer was lower (r = 0.69). The observer scale showed better agreement than the Vancouver scale because the coefficient of variation was lower (18 percent and 22 percent, respectively). The concurrent validity of the observer scale in relation to the Vancouver scale is high (r = 0.89, p < 0.001). Linear regression of the general opinions on scars of the observer and the patient showed that the observer's opinion is influenced by vascularization, thickness, pigmentation, and relief, whereas the patient's opinion is mainly influenced by itching and the thickness of the scar. Such an impact of itching and thickness of the scar on the patient's opinion is an important and novel finding. The Patient and Observer Scar Assessment Scale offers a suitable, reliable, and complete scar evaluation tool.     

Spectroscopic characterization and structural modeling of prolamin from maize and pearl millet

Biophysical methods and structural modeling techniques have been used to characterize the prolamins from maize (Zea mays) and pearl millet (Pennisetum americanum). The alcohol-soluble prolamin from maize, called zein, was extracted using a simple protocol and purified by gel filtration in a 70% ethanol solution. Two protein fractions were purified from seed extracts of pearl millet with molecular weights of 25.5 and 7 kDa, as estimated by SDS-PAGE. The high molecular weight protein corresponds to pennisetin, which has a high -helical content both in solution and the solid state, as demonstrated by circular dichroism and Fourier transform infrared spectra. Fluorescence spectroscopy of both fractions indicated changes in the tryptophan microenvironments with increasing water content of the buffer. Low-resolution envelopes of both fractions were retrieved by ab initio procedures from small-angle X-ray scattering data, which yielded maximum molecular dimensions of about 14 nm and 1 nm for pennisetin and the low molecular weight protein, respectively, and similar values were observed by dynamic light scattering experiments. Furthermore, ¹H nuclear magnetic resonance spectra of zein and pennisetin do not show any signal below 0.9 ppm, which is compatible with more extended solution structures. The molecular models for zein and pennisetin in solution suggest that both proteins have an elongated molecular structure which is approximately a prolate ellipsoid composed of ribbons of folded -helical segments with a length of about 14 nm, resulting in a structure that permits efficient packing within the seed endosperm.     

Gravitropism in Phycomyces: threshold determination on a clinostat centrifuge

The absolute sensitivity of sporangiophores of Phycomyces blakesleeanus to centrifugal acceleration was determined on a clinostat centrifuge. The centrifuge provides centrifugal accelerations ranging from 10(-4) to 6 x g. The rotor of the centrifuge, which accommodates 96 culture vials with single sporangiophores, is clinostatted, that is, turning "head over", at slow speed (1 rev min(-1)) while it is running. The negative gravitropism of sporangiophores is characterized by two components: a polar angle, which is measured in the plane of bending, and an aiming-error angle, which indicates the deviation of the plane of bending from the vector of the centrifugal acceleration. Dose-response curves were generated for both angles with centrifugations lasting 3, 5, and 8 h. The threshold for the polar angle depends on the presence of statoliths, so-called octahedral protein crystals in the vacuoles. The albino strain C171 carAcarR (with crystals) has a threshold near 10(-2) x g while the albino strain C2 carAgeo-3 (without crystals) has a threshold of about 2 x 10(-1) x g. The threshold for the aiming error angle is ill defined and is between 10(-2) and 10(-1) x g. The threshold for the polar angle of the wild type NRRL 1555 (with crystals) is near 8 x 10(-2) x g.     

Evolving nature of the AP2 alpha-appendage hub during clathrin-coated vesicle endocytosis

Clathrin-mediated endocytosis involves the assembly of a network of proteins that select cargo, modify membrane shape and drive invagination, vesicle scission and uncoating. This network is initially assembled around adaptor protein (AP) appendage domains, which are protein interaction hubs. Using crystallography, we show that FxDxF and WVxF peptide motifs from synaptojanin bind to distinct subdomains on alpha-appendages, called 'top' and 'side' sites. Appendages use both these sites to interact with their binding partners in vitro and in vivo. Occupation of both sites simultaneously results in high-affinity reversible interactions with lone appendages (e.g. eps15 and epsin1). Proteins with multiple copies of only one type of motif bind multiple appendages and so will aid adaptor clustering. These clustered alpha(appendage)-hubs have altered properties where they can sample many different binding partners, which in turn can interact with each other and indirectly with clathrin. In the final coated vesicle, most appendage binding partners are absent and thus the functional status of the appendage domain as an interaction hub is temporal and transitory giving directionality to vesicle assembly.