Altered expression of transforming growth factor-βs in chronic renal rejection

Altered expression of transforming growth factor-βs in chronic renal rejection. We examined the altered expression of transforming growth factor-βs in chronic renal rejection in humans, including transforming growth factor beta-1 (TGF-β1), TGF-β2, TGF-β3 and their receptors, transforming growth factor beta receptor type I (TβR-I) and TβR-II. Using Northern blot analysis and immunohistochemistry, 10 specimens of chronically rejected and 8 normal kidney samples were analyzed. By Northern blot analysis the expression of mRNA encoding TGF-β1, TGF-β2, TGF-β3 (P < 0.02), TβR-I and TβR-II (P < 0.02) was decreased in chronically rejected renal cortex samples, compared to normal controls. Immunohistochemical analysis of the normal renal cortex showed strong immunostaining for TGF-β1 and TGF-β3, and mild immunostaining for TGF-β2 in the proximal and distal tubulointerstitium, but no signal for any of the TGF-β isoforms in the glomeruli or in the cortical vessels. In sharp contrast, the glomeruli and the cortical vessels of the rejected kidney specimens exhibited strong immunostaining for TGF-β1 and TGF-β3, whereas the tubules revealed a decrease in immunoreactivity. TβRI and TβRII immunostaining showed similar changes as observed with TGF-β1 and TGF-β3 antibodies. There was a concomitant increase in B-cell accumulation in the glomeruli, while T-cells and macrophages were diffusely abundant in the rejected samples. Since TGF-βs are potent inducers of extracellular matrix proteins and have been shown to be involved in fibrotic disease, the increase in TGF-β1 and TGF-β3 immunoreactivity in the glomeruli suggests that there is a redistribution in TGF-β expression in chronic renal allograft rejection. Together with changes affected by B-cell mediated immunity, the above alterations might contribute to the histopathological changes that occur in this disorder, such as intimal fibrosis, arteriosclerosis and glomerular and tubular sclerosis

High-level secretion of native recombinant human calreticulin in yeast

Additional file 2. Elution profiles of recombinant CRT in anion-exchange chromatography. Separation of CRT from S. cerevisiae (A and B) was performed on a pre-packed 5 ml Q-Sepharose column (GE LifeSciences cat. no. 17-5156-01), equilibrated with 20 mM l-His, pH 5.5, 100 mM NaCl buffer, in 15 column volumes linear gradient from 100 mM to 500 mM NaCl. Separation of CRT from P. pastoris (C and D) was performed on a 15 ml Q-Sepharose sorbent (GE LifeSciences cat. no. 17-0510-10) packed into the XK 16/20 column (GE Lifesciences, cat. no. 28-9889-37), equilibrated with 20 mM l-His, pH 5.5, 100 mM NaCl buffer, in 10 column volumes linear gradient from 100 to 500 mM NaCl. Elution profiles (A and C) and SDS-PAGE gels of corresponding fractions (B and D) are shown. Dashed lines define fractions that were pooled and used for further experiments with purified protein

Differential expression of transforming growth factor-β isoforms and receptors in experimental membranous nephropathy

Transforming growth factor-β1 stimulates matrix production by glomerular mesangial and epithelial cells. In membranous nephropathy (MN) overproduction of matrix by glomerular epithelial cells (GEC) is believed to be responsible for glomerular basement membrane thickening and spikes. We studied experimental MN in rats (passive Heymann nephritis, PHN) at 5, 10 and 30 days. PHN rats exhibited a marked increase in GEC immunostaining for TGF-β2 at all time points. TGF-β3 staining was increased at day 10 only, and TGF-β1 was unchanged. Glomerular mRNA for TGF-β2 and -β3 was increased by day 5 when urine protein increased, whereas TGF-β1 was not. TGF-β2 bioactivity was increased at day 5. There was also a marked increase in GEC immunostaining for TGF-β receptor type I (TβIR) and TGF-β receptor type II (TβIIR) at all time points in PHN. mRNA levels for both receptors increased at day 5. Increases in protein expression and mRNA levels for the TGF-β2 and -β3 isoforms, and TβIR and TβRII were prevented by complement depletion. We conclude that complement-mediated injury to the GEC in vivo is associated with the up-regulation of TGF-β2 and -β3 isoforms, an increase in TGF-β2 bioactivity, and an increase in TβRI and TβRII expression. This contrasts with changes in TGF-β1 reported in mesangial disease, suggesting that TGF-β2 and -β3 may be important in diseases of the GEC. The differential expression of TGF-β isoforms and receptors may be important determinants of the GEC response to injury

Overview of the Role for Calreticulin in the Enhancement of Wound Healing through Multiple Biological Effects

Calreticulin (CRT), an intracellular chaperone protein crucial for the proper folding and transport of proteins through the endoplasmic reticulum, has more recent acclaim as a critical regulator of extracellular functions, particularly in mediating cellular migration and as a requirement for phagocytosis of apoptotic cells. Consistent with these functions, we show that the topical application of CRT has profound effects on the process of wound healing by causing a dose-dependent increase in epithelial migration and granulation tissue formation in both murine and porcine normal and impaired animal models of skin injury. These effects of CRT are substantiated, in vitro, as we show that CRT strongly induces cell migration/wound closure of human keratinocytes and fibroblasts, using a wound/scratch plate assay, and stimulates cellular proliferation of human keratinocytes, fibroblasts, and vascular endothelial cells, providing mechanistic insight into how CRT functions in repair. Similarly, in both animal models, the histology of the wounds show marked proliferation of basal keratinocytes and dermal fibroblasts, dense cellularity of the dermis with notably increased numbers of macrophages and well-organized collagen fibril deposition. Thus, CRT profoundly affects the wound healing process by recruiting cells essential for repair into the wound, stimulating cell growth, and increasing extracellular matrix production

Motor Preparatory Activity in Posterior Parietal Cortex is Modulated by Subjective Absolute Value

For optimal response selection, the consequences associated with behavioral success or failure must be appraised. To determine how monetary consequences influence the neural representations of motor preparation, human brain activity was scanned with fMRI while subjects performed a complex spatial visuomotor task. At the beginning of each trial, reward context cues indicated the potential gain and loss imposed for correct or incorrect trial completion. FMRI-activity in canonical reward structures reflected the expected value related to the context. In contrast, motor preparatory activity in posterior parietal and premotor cortex peaked in high “absolute value” (high gain or loss) conditions: being highest for large gains in subjects who believed they performed well while being highest for large losses in those who believed they performed poorly. These results suggest that the neural activity preceding goal-directed actions incorporates the absolute value of that action, predicated upon subjective, rather than objective, estimates of one's performance

Structure and functional characterization of pyruvate decarboxylase from Gluconacetobacter diazotrophicus

BACKGROUND: Bacterial pyruvate decarboxylases (PDC) are rare. Their role in ethanol production and in bacterially mediated ethanologenic processes has, however, ensured a continued and growing interest. PDCs from Zymomonas mobilis (ZmPDC), Zymobacter palmae (ZpPDC) and Sarcina ventriculi (SvPDC) have been characterized and ZmPDC has been produced successfully in a range of heterologous hosts. PDCs from the Acetobacteraceae and their role in metabolism have not been characterized to the same extent. Examples include Gluconobacter oxydans (GoPDC), G. diazotrophicus (GdPDC) and Acetobacter pasteutrianus (ApPDC). All of these organisms are of commercial importance. RESULTS: This study reports the kinetic characterization and the crystal structure of a PDC from Gluconacetobacter diazotrophicus (GdPDC). Enzyme kinetic analysis indicates a high affinity for pyruvate (KM 0.06 mM at pH 5), high catalytic efficiencies, pHopt of 5.5 and Topt at 45 degrees C. The enzyme is not thermostable (T of 18 minutes at 60 degrees C) and the calculated number of bonds between monomers and dimers do not give clear indications for the relatively lower thermostability compared to other PDCs. The structure is highly similar to those described for Z. mobilis (ZmPDC) and A. pasteurianus PDC (ApPDC) with a rmsd value of 0.57 A for C? when comparing GdPDC to that of ApPDC. Indole-3-pyruvate does not serve as a substrate for the enzyme. Structural differences occur in two loci, involving the regions Thr341 to Thr352 and Asn499 to Asp503. CONCLUSIONS: This is the first study of the PDC from G. diazotrophicus (PAL5) and lays the groundwork for future research into its role in this endosymbiont. The crystal structure of GdPDC indicates the enzyme to be evolutionarily closely related to homologues from Z. mobilis and A. pasteurianus and suggests strong selective pressure to keep the enzyme characteristics in a narrow range. The pH optimum together with reduced thermostability likely reflect the host organisms niche and conditions under which these properties have been naturally selected for. The lack of activity on indole-3-pyruvate excludes this decarboxylase as the enzyme responsible for indole acetic acid production in G. diazotrophicus.IS

Phylogenomic analysis of a 55.1 kb 19-gene dataset resolves a monophyletic Fusarium that includes the Fusarium solani Species Complex

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user¿s needs and established successful practice. In 2013, the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani species complex (FSSC). Subsequently, this concept was challenged in 2015 by one research group who proposed dividing the genus Fusarium into seven genera, including the FSSC described as members of the genus Neocosmospora, with subsequent justification in 2018 based on claims that the 2013 concept of Fusarium is polyphyletic. Here, we test this claim and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a genus Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students, and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species described as genus Neocosmospora were recombined in genus Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural, and practical taxonomic option availabl

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead