Makorin ring zinc finger protein 1 (MKRN1), a novel poly(A)-binding protein-interacting protein, stimulates translation in nerve cells

The poly(A)-binding protein (PABP), a key component of different ribonucleoprotein complexes, plays a crucial role in the control of mRNA translation rates, stability, and subcellular targeting. In this study we identify RING zinc finger protein Makorin 1 (MKRN1), a bona fide RNA-binding protein, as a binding partner of PABP that interacts with PABP in an RNA-independent manner. In rat brain, a so far uncharacterized short MKRN1 isoform, MKRN1-short, predominates and is detected in forebrain nerve cells. In neuronal dendrites, MKRN1-short co-localizes with PABP in granule-like structures, which are morphological correlates of sites of mRNA metabolism. Moreover, in primary rat neurons MKRN1-short associates with dendritically localized mRNAs. When tethered to a reporter mRNA, MKRN1-short significantly enhances reporter protein synthesis. Furthermore, after induction of synaptic plasticity via electrical stimulation of the perforant path in vivo, MKRN1-short specifically accumulates in the activated dendritic lamina, the middle molecular layer of the hippocampal dentate gyrus. Collectively, these data indicate that in mammalian neurons MKRN1-short interacts with PABP to locally control the translation of dendritic mRNAs at synapses.     

Collagen-binding proteins in age-dependent changes in renal collagen turnover: microarray analysis of mRNA expression

Aging is associated with progressive structural and functional deterioration of the kidney. Among the morphological changes associated with renal aging is an accumulation of extracellular matrix (ECM) in the glomeruli and tubuloinsterstitium, which may ultimately lead to the development of renal fibrosis. The mechanisms governing the regulation of ECM metabolism during renal aging are only incompletely defined. We present data from a genome-wide mRNA expression study on renal tissue from 90 wk old male Wistar rats and 10 wk old controls using Illumina BeadArray cDNA microarray. Regulation of candidate gene products was verified by real-time PCR. Morphological changes were evaluated by routine histological methods. Activated fibroblasts were identified by their expression of alpha-smooth muscle actin and collagen I. Morphological analysis demonstrated an expansion of the tubulointerstitial compartment with increased amounts of fibrous collagen but no overt glomerular or tubular damage in the aged rats. Activated fibroblasts were readily detectable in the adventitial layer of large renal vessels in controls and were not found in the old animals. In agreement with this finding, gene expression analysis revealed significant downregulation of collagen I mRNA along with numerous other ECM components. Concomitantly, collagen-stabilizing proteins were induced, whereas matrix metalloproteinase 9, an enzyme involved in collagen breakdown, was reduced. In conclusion, our results suggest that ECM expansion during renal aging results from an augmented stabilization in conjunction with a reduced breakdown of collagen fibers. Collagen stabilizing proteins may be essential for the control of renal ECM turnover and the pathogenesis of kidney fibrosis.     

Protein Brownian Rotation at the Glass Transition Temperature of a Freeze-Concentrated Buffer Probed by Superparamagnetic Nanoparticles

For applications from food science to the freeze-thawing of proteins it is important to understand the often complex freezing behavior of solutions of biomolecules. Here we use a magnetic method to monitor the Brownian rotation of a quasi-spherical cage-shaped protein, apoferritin, approaching the glass transition T_g in a freeze-concentrated buffer (Tris-HCl). The protein incorporates a synthetic magnetic nanoparticle (Co-doped Fe₃O₄ (magnetite)). We use the magnetic signal from the nanoparticles to monitor the protein orientation. As T decreases toward T_g of the buffer solution the protein’s rotational relaxation time increases exponentially, taking values in the range from a few seconds up to thousands of seconds, i.e., orders of magnitude greater than usually accessed, e.g., by NMR. The longest relaxation times measured correspond to estimated viscosities >2 MPa s. As well as being a means to study low-temperature, high-viscosity environments, our method provides evidence that, for the cooling protocol used, the following applies: 1), the concentration of the freeze-concentrated buffer at T_g is independent of its initial concentration; 2), little protein adsorption takes place at the interface between ice and buffer; and 3), the protein is free to rotate even at temperatures as low as 207 K.     

An amphipathic cyclic tetrapeptide scaffold containing halogenated β2,2‐amino acids with activity against multiresistant bacteria

The present study describes the synthesis and biological studies of a small series of head‐to‐tail cyclic tetrapeptides of the general structure c(Lys‐β^2,2‐Xaa‐Lys) containing one lipophilic β^2,2‐amino acid and Lys, Gly, Ala, or Phe as the Xaa residue in the sequence. The peptides were investigated for antimicrobial activity against gram‐positive and gram‐negative reference strains and 30 multiresistant clinical isolates including strains with extended spectrum β‐lactamase—carbapenemase (ESBL‐CARBA) production. Toxicity was determined against human red blood cells. The most potent peptides showed high activity against the gram‐positive clinical isolates with minimum inhibitory concentrations of 4–8 μg/mL and low haemolytic activity. The combination of high antimicrobial activity and low toxicity shows that these cyclic tetrapeptides containing lipophilic β^2,2‐amino acids form a valuable scaffold for designing novel antimicrobial agents.     

Flow cytometric analysis of the chromosomes and stability of a wheat cell-culture line

A rapidly growing, long-term suspension culture derived from Triticum aestivum L. (wheat) was synchronized using hydroxyurea and colchicine, and a chromosome suspension with chromosomes was made. After staining with the DNA-specific fluorochromes Hoechst 33258 and Chromomycin univariate and bivariate flow-cytometry histograms showed 15 clearly resolved peaks corresponding to individual chromosome types or groups of chromosomes with similar DNA contents. The flow karyotype was closely similar to a histogram of DNA content measurements of Feulgen-stained chromosomes made by microdensitometry. We were able to show the stability of the flow karyotype of the cell line over a year, while a parallel subculture had a slightly different, stable, karyotype following different growth conditions. The data indicate that flow cytometric analysis of plant karyotypes enables accurate, statistically precise chromosome classification and karyotyping of cereals. There was little overlap between individual flow-histogram peaks, so the method is useful for flow sorting and the construction of chromosome specific-recombinant DNA libraries. Using bivariate analysis, the AT:GC ratio of all the chromosomes was remarkably similar, in striking contrast to mammalian flow karyotypes. We speculate about a fundamental difference in organization and homogenization of DNA sequences between chromosomes within mammalian and plant genomes. Received: 24 April 1996 / Accepted: 24 May 1996