Hyaluronate synthesized by cultured skin fibroblasts derived from patients with Werner's syndrome.

Hyaluronate in cultured skin fibroblasts derived from patients with Werner's syndrome, who excrete large amounts of urinary hyaluronate, was investigated. The amount of hyaluronate secreted into the medium by Werner's fibroblasts was 2-3-times that of normal fibroblasts, whereas no difference in enzyme activities related to the degradation of hyaluronate was found. Werner's fibroblasts were then cultured in the presence of [3H]glucosamine, and the amount of [3H]hyaluronate and its chain lengths in the medium and matrix (trypsinate) fractions were compared with those of normal cells. No significant difference in the chain length of hyaluronate was observed between normal and Werner's fibroblasts. On the other hand, a significant increase of hyaluronate was found in the matrix fraction of Werner's fibroblasts when the cells reached confluency. In addition, a hyaluronate of small chain length was found in the matrix fraction of Werner's fibroblasts, although this was absent from that of normal cells. It was concluded that the constituents of the extracellular matrix of Werner's fibroblasts differed from those of normal cells, characterized by the presence of a large amount of hyaluronate and a relatively small hyaluronate chain.     

Detection of Macromolecular Fractions in HCN Polymers Using Electrophoretic and Ultrafiltration Techniques

Elucidating the origin of life involves synthetic as well as analytical challenges. Herein, for the first time, we describe the use of gel electrophoresis and ultrafiltration to fractionate HCN polymers. Since the first prebiotic synthesis of adenine by Oró, HCN polymers have gained much interest in studies on the origins of life due to the identification of biomonomers and related compounds within them. Here, we demonstrate that macromolecular fractions with electrophoretic mobility can also be detected within HCN polymers. The migration of polymers under the influence of an electric field depends not only on their sizes (one‐dimensional electrophoresis) but also their different isoelectric points (two‐dimensional electrophoresis, 2‐DE). The same behaviour was observed for several macromolecular fractions detected in HCN polymers. Macromolecular fractions with apparent molecular weights as high as 250 kDa were detected by tricine‐SDS gel electrophoresis. Cationic macromolecular fractions with apparent molecular weights as high as 140 kDa were also detected by 2‐DE. The HCN polymers synthesized were fractionated by ultrafiltration. As a result, the molecular weight distributions of the macromolecular fractions detected in the HCN polymers directly depended on the synthetic conditions used to produce these polymers. The implications of these results for prebiotic chemistry will be discussed.     

Altered frequency of initiation sites of DNA replication in Werner's syndrome cells

Summary DNA replication of cultured fibroblasts of early passage derived from Werner's syndrome (adult progeria) patients and from normal subjects were compared by DNA fiber autoradiography. The frequency of replication initiation was decreased in Werner's syndrome cells derived from five patients compared with that in normal cells derived from three persons of different ages. The rate of DNA chain elongation did not differ between Werner's syndrome cells and normal cells.     

Molecular characteristic of antibodies in the blood serum and in the lymph nodes during fractional immunization with small doses of bacterial vaccines.

An experiment on 58 rabbits demonstrated that only macromolecular agglutinins 19S are present in extracts from the lymph nodes after fractional immunization with small doses of typhoid vaccine or some dysentery vaccine. At the same time, antibodies in the blood serum are distributed in fractions of different molecular weight, starting from the 14th day of experiment. It is very probable that this distribution of antibodies is associated with the occurrence in the blood of an inhibitor of the activity of macromolecular antibodies.     

A mucous secretion in the malpighian tubes of a neotropical bumblebee,Bombus atratus Franklin

Summary A filamentous secretion composed of carboxylated and sulfated acid glycosaminoglycans (AGAG), neutral polysaccharides, and protein(s) appears in the lumen of the Malpighian tubes of the fully grown larvae of the bumblebee,Bombus atratus Franklin. A well-ordered macromolecular array was demonstrated specially for the carboxylated AGAG components of this secretion, based on their linear dichroism and birefringence properties. It is suggested that the carboxylated AGAG macromolecules can acquire a helical conformation when present at the lumen of the organ. The mucous secretion elaborated by the Malpighian tubes ofB. atratus is excreted from the larvae in the form of condensed filaments. Its function remains unclear. Globules with concentric lamination containing protein and neutral polysaccharides were seen detaching from the apical border of the epithelial cells of the Malpighian tubes of the fully grown larvae. It could not be established whether they contribute for the elaboration of the filamentous secretion. Morphologically similar globules have been assumed in some other insect groups to originate from cytolysomes. Urate crystals surrounded by a halo of calcium granules were also found in the lumen of the Malpighian tubes ofB. atratus.     

Matrix-assisted ultraviolet laser-desorption ionization time-of-flight mass spectrometry of sulfated mannans from the red seaweed Nothogenia fastigiata

Matrix-assisted ultraviolet laser-desorption ionization time-of-flight mass spectrometry (UV-MALDI-TOF-MS) was applied to sulfated xylo-mannan fractions from Nothogenia fastigiata in order to determine their molecular weights and distribution profiles. The number-average molecular weight calculated from the spectra was similar to that determined by chemical end-group analysis for the lower molecular weight fractions. For the other fractions, the number-average molecular weight was lower than that chemically determined; the increased difference may be attributed to higher desorption difficulties and, consequently, mass-dependent discrimination. A reconstructed spectrum, using the peaks obtained from all the fractions, suggested an unimodal distribution. The best results were obtained by using 2,5-dihydroxybenzoic acid as matrix doped with 1-hydroxyisoquinoline and with harmane and nor-harmane.     

Effects of radical-scavenging enzymes and reduced oxygen exposure on growth and chromosome abnormalities of Werner syndrome cultured skin fibroblasts

Summary Two sister strains of skin fibroblast-like (FL) cells from a patient with Werner's syndrome (adult progeria) were grown in regular tissue culture medium or medium supplemented with the radical-scavenging enzymes superoxide dismutase and catalase. Differences in proliferative potential were shown to be due to strain variation rather than to treatment with the enzymes. Two independent strains were also grown in ambient (18%) or reduced (1%) concentrations of oxygen. All cultures (untreated, treated with enzymes, treated with reduced oxygen exposure) displayed the limited in vitro growth potential and cytogenetic abnormality characteristic of Werner's syndrome FL cells; thus the underlying defect in Werner's syndrome does not appear to be related to defective protection against or repair of damage by free radicals.     

Clonal structural chromosomal rearrangements in lymphocytes of four patients with Werner's syndrome

Multiple numerical and structural chromosome abnormalities were found in cultured lymphocytes of four patients with Werner's syndrome. The proportion of metaphases with structural and/or numerical aberrations varied from 30 to 44% and several of them were clonal. These results confirm definitively that Werner's syndrome is a chromosome rearrangement syndrome and that these non-constitutional chromosome changes are not exclusive of cultured fibroblasts but present also in lymphocytes.     

Elevation of urinary hyaluronic acid in Werner's syndrome and progeria

Werner's syndrome and Hutchinson-Gilford progeria syndrome (progeria) are human genetic diseases which may serve as models for the study of premature aging. The basic defects underlying these diseases are unknown. An abnormally high level of urinary hyaluronic acid (HA) excretion has been previously reported in several Werner's and one progeria subject, all from Japan. To determine if a high HA level is a reliable marker for these diseases, we quantitated the urinary excretion of HA in three progeria subjects, one subject with an atypical progeroid syndrome, and a Werner's syndrome subject. Compared to controls, the total urinary HA was found to be markedly increased in the three progeria samples and in the Werner's syndrome sample. These findings support the previous observations indicating elevated HA may be a specific marker for these diseases.     

Premature aging in RecQ helicase-deficient human syndromes

The RecQ family of DNA helicases have potential roles in DNA repair, replication and/or recombination pathways. In humans, a defect in the RecQ family helicases encoded by the BLM, WRN and RECQ4 genes gives rise to Bloom's (BS), Werner's (WS) and Rothmund-Thomson (RTS) syndromes, respectively. These disorders are associated with cancer predisposition and/or premature aging. In Bloom's syndrome, affected individuals are predisposed to many types of cancer at an early age. Werner's syndrome is a premature aging disorder with a complex phenotype, which includes many age-related disorders that develop from puberty, including greying and thinning of the hair, bilateral cataract formation, type II diabetes mellitus, osteoporosis and atherosclerosis. The phenotype of Rothmund-Thomson syndrome patients also consists of some features associated with premature aging, as well as predispositon to certain cancers. Here, we discuss the molecular basis of these RecQ helicase-deficient disorders.     

Enhanced ability of heparin-carrying polystyrene (HCPS) to bind to heparin-binding growth factors and to inhibit growth factor-induced endothelial cell growth

Heparin-carrying polystyrene (HCPS) consists of low-molecular-weight heparin chains enriched in trisulfated disaccharide structures linked to a polystyrene core. In this study, the interactions between HCPSs of various molecular weights and heparin-binding growth factors, VEGF(165), FGF-2, and HGF, were compared to the interactions of the same factors with native heparin, periodate-oxidized heparin (IO(4)-heparin) and periodate-oxidized alkaline-degraded heparin (IO(4)-LMW-heparin). The binding of each growth factor to heparin-agarose beads (heparin-beads) was more strongly inhibited by HCPSs in a molecular weight-dependent manner than by native heparin or the modified heparins, indicating a stronger interaction between HCPS and these growth factors. HCPSs also inhibit heparin-binding growth factor-induced endothelial cell growth in a molecular weight-dependent manner much more strongly than the native or modified heparins. However, HCPSs did not inhibit the mitogenic activity of VEGF(121), which has a non-heparin-binding nature. Thus, HCPSs exhibit enhanced abilities to interact with each of the heparin-binding growth factors studied and to inhibit heparin-binding growth factor-induced endothelial cell proliferation in a molecular weight-dependent manner. These effects might be ascribed to the heparin-clustering effect of HCPSs.     

Werner's syndrome: A review of recent research with an analysis of connective tissue metabolism,growth control of cultured cells,and chromosomal aberrations

Summary Werner's syndrome is a rare, autosomal recessive condition with multiple progeroid features, but it is an imitation of aging rather than accelerated or premature senescence. Somatic chromosome aberrations occur in multipe tissues in vivo and in vitro, and there is an increased incidence of neoplasia. Thus, Werner's syndrome can be classified in the group of chromosome instability syndromes. Recent findings provide additional support for the concept that there is aberration of connective tissue metabolism in Werner's syndrome, but it is unclear whether this is a primary or secondary manifestation of the underlying genetic defect. Abnormal growth characteristics are observed in cultured skin fibroblast-like cells and this provides another avenue for current research. Identification of the basic genetic defect in Werner's syndrome might clarify our understanding of the normal aging process in general, or might elucidate specific aspects such as the development of neoplasia, atherosclerosis, diabetes, or osteoporosis.     

Effects of amylopectin structure and molecular weight on microstructural and rheological properties of mixed beta-lactoglobulin gels

Nongelling amylopectin fractions from potato and barley have been used to form mixed beta-lactoglobulin gels. The amylopectin fractions were produced by varying the time of alpha-amylase hydrolysis followed by sequential ethanol precipitation. The molecular weights, radius of gyration, chain length distribution, and viscosity of the fractions were established. The mixed gels were analyzed rheologically with dynamic mechanical analysis in shear and microstructurally with light microscopy, transmission electron microscopy, and nuclear magnetic resonance spectroscopy. The result of the gel studies clearly showed that small differences in the molecular weight of amylopectins have a significant influence on the kinetics of protein aggregation and thereby on the gel microstructure and the rheological behavior of the gel. Both an increase in the molecular weight and a higher concentration of amylopectins resulted in a more open protein network structure, with thicker strands of larger and more close-packed beta-lactoglobulin clusters, which showed a larger storage modulus. The transmission electron micrographs revealed that degraded amylopectins were enclosed inside the protein clusters in the mixed gels, whereas nondegraded amylopectin was only found outside the protein clusters. The volume-weighted mean value of the molecular weight of the amylopectins was found to vary between 3.2 x 10(4) and 5.0 x 10(7) Da and the ratio of gyration between 14 and 61 nm. The maximum in chain length distribution was generally somewhat distributed toward longer chain lengths for potato compared to barley, but the differences in chain length distribution were minor compared to those seen in the molecular weight and ratio of gyration between the fractions.     

Structural subunits of chondroitin sulfate isomers isolated from different aged human kidneys

Acidic glycosaminoglycans (AGAG) were extracted from whole human kidney tissues of different aged subjects and were purified by precipitation with cetylpyridinium chloride and by an ion exchange resin column chromatography. The prepared AGAG were individually digested with chondroitinase-AC and they were further digested with chondroitinase-ABC. The resulting unsaturated disaccharides and the undigested AGAG were identified by paper chromatography, electrophoresis, and other chemical analyses. The AGAG, which were resistant to chondroitinase-AC, were mainly heparan sulfates and their proportion to total AGAG appeared to decrease with the advance of age. Dermatan sulfate plus oversulfated dermatan sulfate were composed of one-fifth to one-third of the AGAG resistant to chondroitinase-AC, and their proportion tended to increase with the advance of age.     

Clonal structural chromosomal rearrangements in primary fibroblast cultures and in lymphocytes of patients with Werner's syndrome

Summary The cytogenetics of six cases of adult progeria (Werner's syndrome) from three Sardinian families were investigated. The overall increased incidence of chromosome breakage found in cultured lymphocytes and fibroblasts seems to be age-dependent. The occurrence of clonal variegated translocation mosaicism, previously found by other authors in fibroblast cell lines derived from Werner patients was demonstrated also in fibroblasts analyzed in situ on the outgrowth halos from primary skin explants; a strong indication that these aberrations are present in the in vivo precursors. The same type of clonal structural aberration was found for the first time also in 72h-cultured lymphocytes. These findings demonstrate that Werner's syndrome is indeed a further example of a chromosome rearrangement syndrome.     

汞与硒在不同汞暴露水平地区猪肝脏和肾脏蛋白质中的分布

通过对贵州万山汞污染地区及北京地区猪肝脏和肾脏组织上清液进行凝胶过滤色谱分离(SephadexG 10 0 ) ,随后用原子荧光法测定它们蛋白质组分中汞和硒的含量 ,研究在汞暴露水平不同状态下微量元素汞和硒在动物体蛋白质分子水平上的分布 .发现这两个地区猪肝脏和肾脏组织上清液蛋白质组分中汞和硒的分布模式有明显差异 .贵州万山汞污染地区猪肝脏上清液中汞浓度比北京地区高 ,硒浓度也相应高 ,且前者与高分子量和低分子量蛋白结合的硒均明显高于后者 ;而北京地区猪肝脏上清液中的硒主要以与高分子量蛋白结合的形式存在 .贵州汞污染地区猪肝脏上清液中汞主要与高分子量蛋白结合 ,而北京地区猪肝脏上清液中汞则分布较为均匀 .贵州万山地区猪肾脏上清液中 ,含硒峰在高分子量蛋白区和低分子量区都有分布 ;而北京地区猪肾脏上清液中 ,硒则主要集中分布于高分子量蛋白范围 .这两个地区猪肾脏上清液中都有分子量约为 11kD的金属硫蛋白 (MT)存在 ,北京地区猪肾脏上清液中汞主要以与金属硫蛋白结合的形式出现 ,而贵州万山地区猪肾脏上清液中的汞除与金属硫蛋白结合外 ,尚有相当大部分是以与高分子量蛋白结合的形式存在 .研究结果表明 ,由于这两个地区汞暴露水平的差异 ,不仅使这两地区猪肝、肾上清液中的汞与硒含量     

Mutagen sensitivity and the repair process of the lymphocytes in the Werner syndrome

In this study, we analyzed the cell cycle kinetics, the radiosensitivity, the repair process and the induction of sister chromatid exchanges in lymphocytes from the Werner's syndrome. When compared to a normal population, no statistical significant differences were observed for the cell cycle kinetics and the radiosensitivity. However, differences exist with respect to the repair process and lymphocytes from the Werner's syndrome are much more sensitive to the induction of SCE's.     

Comprehensive peptidome analysis of mouse livers by size exclusion chromatography prefractionation and nanoLC-MS/MS identification

Peptidome analysis has received increasing attention in recent years. Cancer diagnosis by serum peptidome has also been reported by peptides' profiling for discovery of peptide biomarkers. Tissue, which may have a higher biomarker concentration than blood, has not been investigated extensively by means of peptidome analysis. Here, a method for the peptidome analysis of mouse liver was developed by the combination of size exclusion chromatography (SEC) prefractionation with nano-liquid chromatography-tamdem mass spectrometry (nanoLC-MS/MS) analysis. The extracted peptides from mouse liver were separated according to their molecular weight using a size exclusion column. MALDI-TOF MS was used to characterize the molecular weight distribution of the peptides in fractions eluted from the SEC column. The low molecular weight (LMW) (MW < 3000 Da) peptides in the collected fractions were directly analyzed by LC-MS/MS which resulted in the identification of 1181 unique peptides (from 371 proteins). The high molecular weight (HMW) (MW > 3000 Da) peptides in the early two fractions from the SEC column were first digested with trypsin, and the resulted digests were then analyzed by LC-MS/MS, which led to the identification of 123 and 127 progenitor proteins of the HMW peptides in fractions 1 and 2, respectively. Analysis of the peptides' cleavage sites showed that the peptides are cleaved in regulation, which may reflect the protease activity and distribution in body, and also represent the biological state of the tissue and provide a fresh source for biomarker discovery.     

THE SUBCELLULAR LOCALIZATION OF ACETYLCHOLINESTERASE AND ITS MOLECULAR FORMS IN PIG CEREBRAL CORTEX

Abstract— The distribution of acetylcholinesterase among the subcellular fractions of pig cerebral cortex was determined. The crude mitochondrial and microsomal fractions obtained by differential centrifugation accounted for 75% of the enzyme, with the remainder divided between the crude nuclear and soluble fractions.
The occurrence and distribution of the multiple molecular forms of AChE was the same in all four fractions with the dominant species of molecular weights 350,000, 270,000 and 60,000. Further purification of the mitochondrial fraction by density gradient centrifugation gave a series of membrane fractions with very similar multiple forms. The one possible exception was the fraction containing the purified synaptosomal membranes where one band of mol wt 270,000 predominated, although the other molecular weight entities were present. The electrophoretic pattern of AChE present in the fractionated microsomes was the same as in the crude preparation. The content and pattern of the multiple molecular forms of AChE was therefore the same in all fractions of pig brain, apart from that containing the purified synaptosomal membranes.     

Mechanical Properties of Mammalian and Fish Gelatins as a Function of the Contents of α-Chain, β-Chain,and Low and High Molecular Weight Fractions

Small-strain oscillatory measurements and size-exclusion chromatography coupled to multiangle laser light scattering were used to study the mechanical properties and the molecular weight distribution, respectively, of acid porcine skin gelatins (type A), lime bovine bone gelatins (type B), and cold water fish gelatins, while principal component analysis (PCA) and partial least squares regression were used to relate the mechanical properties with the molecular weight distribution. The present study suggests a linear relationship between the mechanical properties and the fractions of low molecular weight (LMW) molecules, α-chains, β-chains, and high molecular weight (HMW) molecules. The Bloom value for mammalian gelatin was positively correlated with the fractions of α-chains, β-chains, and HMW molecules and negatively correlated with the fraction of LMW molecules. The dynamic storage modulus for cold water fish gelatin was positively correlated with the fractions of β-chains and HMW molecules and negatively correlated with the fractions of LMW molecules and α-chains.