A novel role of vimentin filaments: binding and stabilization of collagen mRNAs

The stem-loop in the 5' untranslated region (UTR) of collagen α1(I) and α2(I) mRNAs (5'SL) is the key element regulating their stability and translation. Stabilization of collagen mRNAs is the predominant mechanism for high collagen expression in fibrosis. LARP6 binds the 5'SL of α1(I) and α2(I) mRNAs with high affinity. Here, we report that vimentin filaments associate with collagen mRNAs in a 5'SL- and LARP6-dependent manner and stabilize collagen mRNAs. LARP6 interacts with vimentin filaments through its La domain and colocalizes with the filaments in vivo. Knockdown of LARP6 by small interfering RNA (siRNA) or mutation of the 5'SL abrogates the interaction of collagen mRNAs with vimentin filaments. Vimentin knockout fibroblasts produce reduced amounts of type I collagen due to decreased stability of collagen α1(I) and α2(I) mRNAs. Disruption of vimentin filaments using a drug or by expression of dominant-negative desmin reduces type I collagen expression, primarily due to decreased stability of collagen mRNAs. RNA fluorescence in situ hybridization (FISH) experiments show that collagen α1(I) and α2(I) mRNAs are associated with vimentin filaments in vivo. Thus, vimentin filaments may play a role in the development of tissue fibrosis by stabilizing collagen mRNAs. This finding will serve as a rationale for targeting vimentin in the development of novel antifibrotic therapies.     

Ultrasound effect on physical properties of corn starch

High power ultrasound (HPU) represents a non-thermal processing method that has been rapidly researched and used in the last 10 years. The application of power ultrasound offers the opportunity to modify and improve some technologically important compounds which are often used in food products. One of them is starch. The aim of this research was to examine the effect of the high power ultrasound of 24 kHz frequency on rheological and some physical properties of corn starch. Various ultrasound treatments were used; an ultrasound probe set with different intensities (34, 55, 73 W cm⁻²) and treatment times (15 and 30 min) and ultrasound bath of 2 W cm⁻² intensity and treatment times (15 and 30 min). Rheological parameters, turbidity and swelling power of corn starch suspensions were determined for native and ultrasonically treated corn starch suspensions. Differential scanning calorimetry was used in order to examine the pasting properties of corn starch. The results have shown that the ultrasound treatment of corn starch distorts the crystalline region in starch granules. The results of differential scanning calorimetry measurements have shown a decrease in enthalpy of gelatinization. A significant decrease in consistency coefficient (k) has also been observed. The consistency coefficient decreases stepwise jointly with the increasing ultrasound power. The increase in the swelling power is associated with water absorption capacity and corn starch granules solubility, respectively.     

Influence of surgery onto the appearance of the hip joint periarticular calcification in patients with the acetabular fracture

The intention of this study was to investigate the influence of surgery onto the appearance of the hip joint periarticular calcification in different groups of patients who suffered the acetabular fracture. A series of 103 patients with the acetabular fracture was analysed in a retrospective case-control study during a seven-year period. The patients were divided into two groups. The case group was comprised of 21 patients who were operated on due to the posterior acetabular wall fracture, while the control group was formed from 82 patients who underwent conservative treatment and who suffered complex acetabular fracture. To obtain the results, the rate and grade of the hip joint post-injury periarticular calcification formation were observed and analysed. They were considerably lower in patients from the case group who were operated on, compared to patients from the control group where the hip joint arthrosis was more common. In conclusion, considering the results of this paper, the rate and grade of the hip joint osteodegenerative changes may be highly decreased by surgery in patients who sustained the acetabular fracture.     

Analysis of airborne pollen time series originating from Hirst-type volumetric samplers—comparison between mobile sampling head oriented toward wind direction and fixed sampling head with two-layered inlet

Aerobiologia - In this study, we have compared time series of pollen concentration originating from two Hirst-type volumetric samplers that were equipped with different sampling heads. To collect...     

Selective Irreversible Inactivation of Replicating Mengovirus by Nucleoside Analogues: a New Form of Viral Interference

We describe the selective irreversible inhibition of mengovirus growth in cultured cells by a combination of two pyrrolopyrimidine nucleoside analogues, 5-bromotubercidin (BrTu) and tubercidin (Tu). At a concentration of 5 microgram/ml, BrTu reversibly blocked the synthesis of cellular mRNA and rRNA but did not inhibit either mengovirus RNA synthesis or multiplication. BrTu is a potent inhibitor of adenosine kinase, and low concentrations of BrTu (e.g., 0.5 microgram/ml), which did not by themselves inhibit cell growth, blocked phosphorylation of Tu and thus protected uninfected cells against irreversible cytotoxicity resulting from Tu incorporation into nucleic acids. In contrast, in mengovirus-infected cells, BrTu did not completely inhibit Tu incorporation into mengovirus RNA, allowing the formation of Tu-containing functionally defective polynucleotides that aborted the virus development cycle. This increased incorporation of Tu coupled to mengovirus infection could be attributed either to a reduction in the inhibitory action of BrTu and/or its nucleotide derivatives at the level of nucleoside and nucleotide kinases and/or, perhaps, to an effect upon the nucleoside transport system. The virus life cycle in nucleoside-treated cells progressed to the point of synthesis of negative strands and probably to the production of a few defective new positive strands. Irreversible virus growth arrest was achieved if the nucleoside mixture of BrTu (0.5 to 10 microgram/ml) and Tu (1 to 20 microgram/ml) was added no later than 30 min after virus infection and maintained for periods of 2 to 8 h. The cultures thus "cured" of mengovirus infection could be maintained and transferred for several weeks, during which they neither produced detectable virus nor showed a visible cytopathic effect; however, the infected and cured cells themselves, while metabolically viable, were permanently impaired in RNA synthesis and unable to divide. Although completely resistant to superinfecting picornaviruses, they retained the ability to support the growth of several other viruses (vaccinia virus, reovirus, and vesicular stomatitis virus), showing that cured cells had, in general, retained the metabolic and structural machinery needed for virus production. The resistance of cured cells to superinfection with picornaviruses seemed attributable neither to interferon action nor to destruction or blockade of virus receptors but more likely to the consumption of some host factor(s) involved in the expression of early viral functions during the original infection.     

Increase of sensitivity of sputum cytology using high-resolution image cytometry: field study results

Lung cancer remains the leading cause of cancer deaths in the developed world. There is no widely accepted method to screen for this cancer. The most commonly used method remains conventional sputum cytology, but this method is hampered by low sensitivity. We tested the hypothesis that sensitivity of sputum cytology for early lung cancer can be greatly improved by using image analysis of sputum cells, at a modest reduction of specificity.The study was double-blinded and used sputum samples from subjects with well-characterized clinical diagnoses. There were 177 cancers, 98 dysplasias, and 558 normals. The study samples were separated into two independent sets: training set and test set. Sputum samples were collected prospectively from subjects with a high probability of having lung cancer. Seven institutions from five countries participated in the study. All subjects had complete clinical diagnoses which included, as a minimum, negative chest x-rays for all negative cancers, while all cancers had confirmed tissue pathology. Samples were prepared according to the Saccomanno method. For conventional cytology, slides were stained using Papanicolaou stain. For image analysis, slides were stained using a DNA-specific (Feulgen-Thionin) stain. An automated, high-resolution image cytometer was used for measurements.At 90% specificity, sensitivity of 60% can be achieved for adenocarcinoma, compared to only 14% sensitivity of conventional cytology (at 99% specificity). Similarly, 45% sensitivity at 90% specificity can be reached for stages 0 and I lung cancer, compared to only 14% (at 99% specificity) using conventional cytology.Cytometry combined with conventional cytology shows an increase in sensitivity to early-stage cancer and to adenocarcinomas compared to conventional cytology alone. While the results are encouraging, the sensitivity to detect early lung cancer should be further improved to 70-80% at 90-95% specificity before this test can be considered for screening of high-risk individuals for lung cancer. Cytometry (Clin. Cytometry) 50:168-176, 2002.     

Formation of noncanonical DNA structures mediated by human ORC4, a protein component of the origin recognition complex

Many genomic sequences, DNA replication origins included, contain specific structural motifs prone to alternative base pairing. Structural rearrangements of DNA require specific environmental conditions and could be favored by chemical agents or proteins. To improve our understanding of alternative conformations of origins and the manner in which they form, we have investigated the effect of DNA-binding, AAA+ protein human ORC4 on single-stranded origin DNA or various oligonucleotides. Here we demonstrate that human ORC4 stimulated formation of inter- and intramolecular T.A.T triplexes and created novel structures, such as homoadenine duplexes. Adenine-based structures were held together by Hoogsteen hydrogen bonds, as demonstrated on 7-deaza-dAMP- or dAMP-containing substrates, and characterized by increased thermal stability. Adenine pairing occurred only in the presence of human ORC4, in a neutral buffer supplemented with ATP and Mg (2+) ions. The protein mutant that could not bind ATP was inactive in this reaction. Since the action of human ORC4 could be biologically important, its potential impact on DNA replication is discussed.     

Replication slippage versus point mutation rates in short tandem repeats of the human genome

Short tandem repeats (STRs) are subjected to two kinds of mutational modifications: point mutations and replication slippages. The latter is found to be the more frequent cause of STR modifications, but a satisfactory quantitative measure of the ratio of the two processes has yet to be determined. The comparison of entire genome sequences of closely enough related species enables one to obtain sufficient statistics by counting the differences in the STR regions. We analyzed human–chimpanzee DNA sequence alignments to obtain the counts of point mutations and replication slippage modifications. The results were compared with the results of a computer simulation, and the parameters quantifying the replication slippage probability as well as the probabilities of point mutations within the repeats were determined. It was found that within the STRs with repeated units consisting of one, two or three nucleotides, point mutations occur approximately twice as frequently as one would expect on the basis of the 1.2% difference between the human and chimpanzee genomes. As expected, the replication slippage probability is negligible below a 10-bp threshold and grows above this level. The replication slippage events outnumber the point mutations by one or two orders of magnitude, but are still lower by one order of magnitude relative to the mutability of the markers that are used for genotyping purposes.     

Gamma-aminobutyric acid type B receptors are constitutively internalized via the clathrin-dependent pathway and targeted to lysosomes for degradation

Receptor internalization is recognized as an important mechanism for rapidly regulating cell surface numbers of receptors. However, there are conflicting results on the existence of rapid endocytosis of gamma-aminobutyric acid, type B (GABAB) receptors. Therefore, we analyzed internalization of GABAB receptors expressed in HEK 293 cells qualitatively and quantitatively using immunocytochemical, cell surface enzyme-linked immunosorbent assay, and biotinylation methods. The data indicate the existence of rapid constitutive receptor internalization, with the first endocytosed receptors being observed in proximity of the plasma membrane after 10 min. After 120 min, a loss of about 40-50% of cell surface receptors was detected. Stimulation of GABAB receptors with GABA or baclofen did not enhance endocytosis of receptors, indicating the lack of agonist-induced internalization. The data suggest that GABAB receptors were endocytosed via the classical dynamin- and clathrin-dependent pathway and accumulated in an endosomal sorting compartment before being targeted to lysosomes for degradation. No evidence for recycling of receptors back to the cell surface was found. In conclusion, the results indicate the presence of constitutive internalization of GABAB receptors via clathrin-coated pits, which resulted in lysosomal degradation of the receptors.