Calcification inhibitors in rat and human serum and plasma

Rat and human serum and plasma were shown to contain considerable amounts of calcium phosphate precipitation inhibitors. Two general classes of inhibiting molecules were observed for both species: high molecular weight (approx. 30 000-200 000) and low molecular weight (less than 1000). The high molecular weight components eluted from a Bio-Gel P-200 column in two peaks, one at approx. 158 000 and a broader peak at approx. 43 000. The identity of these inhibitors is unknown at present. Low molecular weight inhibitors include magnesium, pyrophosphate, and citrate ions and at least one unidentified component that coelutes with pyrophosphate and citrate on a Bio-Gel P-4 column. Quantitatively, most of the inhibitor activity resides in the high molecular weight components and it is possible that it is this activity which is responsible for maintaining the metastability of the circulating fluids. The role of the low molecular weight components may be to regulate calcification at sites inaccessible to high molecular weight molecules.     

Molecular biology in biorheology

This presentation is aimed at giving some background information on molecular biology, thus serving as an introduction to the Symposium on Molecular Biorheology held during the Sixth International Congress of Biorheology in Vancouver. The papers presented at this Symposium indicate that the use of molecular biological techniques allows the understanding of normal and abnormal rheological properties of cells and organs at the molecular level. It is hoped that these examples will provide an impetus for us to open new frontiers of research in biorheology by taking advantage of the powerful tools developed from recent advances in molecular biology.     

超声分子成像的研究进展

超声成像无创、无放射性、低成本、实时成像的优点,使其成为目前世界上应用最广的成像手段之一。特别是超声造影剂引入之后,超声成像的图像分辨率和灵敏度得到了大大提高,使超声成像在临床上得到了进一步应用。近年来,随着分子生物学和超声成像技术的不断发展,人们提出了"超声分子成像"的概念。它是一项结合了分子靶向造影剂和超声影像技术的能在分子水平下观察病理变化的新兴技术,目前这一技术还处于研究初期阶段。但大量临床前的研究成果已表明超声分子成像在诊断血管生成、炎症和血栓三种疾病具有很大应用前景。本文主要综述了目前常用超声造影剂的种类以及超声分子成像技术的研究现状,并对该技术进行了讨论和展望。     

超声分子成像的研究进展

超声成像无创、无放射性、低成本、实时成像的优点,使其成为目前世界上应用最广的成像手段之一。特别是超声造影剂引入之后,超声成像的图像分辨率和灵敏度得到了大大提高,使超声成像在临床上得到了进一步应用。近年来,随着分子生物学和超声成像技术的不断发展,人们提出了＂超声分子成像＂的概念。它是一项结合了分子靶向造影剂和超声影像技术的能在分子水平下观察病理变化的新兴技术,目前这一技术还处于研究初期阶段。但大量临床前的研究成果已表明超声分子成像在诊断血管生成、炎症和血栓三种疾病具有很大应用前景。本文主要综述了目前常用超声造影剂的种类以及超声分子成像技术的研究现状,并对该技术进行了讨论和展望。     

Yeast RNA partition accompanying adsorption in salt/PEG two-phase system

Summary Partitioning of yeast total RNA in a salt/PEG two-phase system, i.e., a potassium phosphate/PEG system and a ammonium sulfate/PEG system, was characterized with regard to the dependence on the molecular weights of PEG and RNA. The shift in RNA partitioning was investigated for a PEG molecular weight range from 300 to 20000. RNA was partitioned mainly to the top phase in the system with PEG of a molecular weight up to 1000, mainly at the interface or almost equally to both phases in the system with PEG of a molecular weight 1000–2000, and mainly to the bottom phase in the system with PEG of more than 2000 in a molecular weight . The effect of PEG molecular weight on partitioning of low molecular weight RNA, less than 5.8S molecule, was qualitatively similar to that of high molecular weight RNA, more than 17S molecule. However, partitioning of high molecular weight RNA was more one-sided than that of low molecular weight RNA. In the system with PEG1000–2000, remarkable adsorption of high molecular weight RNA at the interface was investigated; more than 90% of the high molecular weight RNA added was concentrated. Adsorption of RNA at the interface was quantitatively demonstrated as a novel example of adsorption of a soluble macromolecule in an aqueous two-phase system.     

The transmembrane proteins in the plasma membrane of normal human erythrocytes. Evaluation employing lactoperoxidase and proteases.

The molecular architecture of the human erythrocyte membrane has been probed using lactoperoxidase-catalyzed iodination in conjunction with Pronase hydrolysis. Resealed, hemoglobin-free ghosts were labeled at the cytoplasmic surface and the external membrane surface was subsequently digested with Pronase. Changes in size of the components labeled at the cytoplasmic surface were readily detected by sodium dodecyl sulfate gel electrophoresis. The protein 3 molecular weight class labeled at the cytoplasmic surface was extensively hydrolyzed at the external surface to produce a major 65000 molecular weight fragment and a minor 45000 molecular weight fragment. When resealed membranes were labeled on the external surface the same 65000 molecular weight labeled component is produced. These results unequivocally demonstrate that the same polypeptides in the protein 3 molecular weight class that can be labeled by lactoperoxidase at the cytoplasmic membrane surface are digested by Pronase at the external surface and are, therefore, transmembrane components. Where it is possible to label one surface of a membrane with lactoperoxidase and reseal the membrane this procedure represents an alternate method for establishing transmembrane configuration of membrane proteins.     

Preliminary characterization of multiple hyaluronidase forms in boar reproductive tract

Hyaluronidases play an important role in gamete interaction and fertility in mammals. The objectives of the present study were to investigate multiple forms of the enzyme in boar reproductive tract using electrophoretic methods. Two forms of hyaluronidase (EC 3.2.1.35) were detected in boar seminal plasma (relative molecular masses of 55,000 and 65,000) using hyaluronic acid-substrate polyacrylamide gel electrophoresis in the presence of SDS. These two forms can be separated by means of affinity chromatography on Heparin-Sepharose. They differ, besides their affinity to heparin, also in the pH optimum of their enzymatic activity. The form with relative molecular mass of 55,000 was active both at the acidic (pH 3.7) and the neutral pH (pH 7.4) and was bound to immobilized heparin. The second form (relative molecular mass 65,000) was active only at acidic pH and did not interact with heparin. The same acidic-active form (65,000) was found in seminal vesicle fluids. The hyaluronidase form which is active both at the acidic and the neutral pH (51,000) was detected in epididymal fluid. In the detergent extracts of boar sperm, three active forms of the enzyme were found (relative molecular masses 55,000, 70,000 and 80,000). The form of relative molecular mass 55,000 was active in a wide range of pH (pH 3-8). The forms of relative molecular masses 70,000 and 80,000 were active only at neutral pH.     

New chromosome 11p15 epigenotypes identified in male monozygotic twins with Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome demonstrating heterogeneous molecular alterations of two imprinted domains on chromosome 11p15. The most common molecular alterations include loss of methylation at the proximal imprinting center, IC2, paternal uniparental disomy (UPD) of chromosome 11p15 and hypermethylation at the distal imprinting center, IC1. An increased incidence of female monozygotic twins discordant for BWS has been reported. The molecular basis for eleven such female twin pairs has been demonstrated to be a loss of methylation at IC2, whereas only one male monozygotic twin pair has been reported with this molecular defect. We report here two new pairs of male monozygotic twins. One pair is discordant for BWS; the affected twin exhibits paternal UPD for chromosome 11p15 whereas the unaffected twin does not. The second male twin pair is concordant for BWS and both twins of the pair demonstrate hypermethylation at IC1. Thus, this report expands the known molecular etiologies for BWS twins. Interestingly, these findings demonstrate a new epigenotype-phenotype correlation in BWS twins. That is, while female monozygotic twins with BWS are likely to show loss of imprinting at IC2, male monozygotic twins with BWS reflect the molecular heterogeneity seen in BWS singletons. These data underscore the need for molecular testing in BWS twins, especially in view of the known differences among 11p15 epigenotypes with respect to tumor risk.     

Continuous Changes in Triacylglycerol Molecular Species Composition by Fatty Acids in Porcine Adipocytes

It has been demonstrated that the composition of molecular species of adipose tissue triacylglycerols (TGs) from farm animals are not equally synthesized and that some molecular species are preferentially synthesized. The objective of the present study was to determine whether exogenous fatty acids (FAs) would affect the TG composition. To this end, the composition of TG molecular species stored in porcine adipocytes differentiated with several long-chain FAs was analyzed by gas chromatography. The addition of each FA for 6 d increased TG molecular species having two or three added FAs. However, the molecular species compositions at 15 d after the addition of each FA resembled those of cells with no added FAs. Moreover, some common molecular species in all experimental cells increased, as well as cells with no added FAs. It was concluded that the addition of FAs increases the contents of specific molecular species, but does not affect the synthetic processes of individual TG molecular species.     

Continuous changes in triacylglycerol molecular species composition by fatty acids in porcine adipocytes

It has been demonstrated that the composition of molecular species of adipose tissue triacylglycerols (TGs) from farm animals are not equally synthesized and that some molecular species are preferentially synthesized. The objective of the present study was to determine whether exogenous fatty acids (FAs) would affect the TG composition. To this end, the composition of TG molecular species stored in porcine adipocytes differentiated with several long-chain FAs was analyzed by gas chromatography. The addition of each FA for 6 d increased TG molecular species having two or three added FAs. However, the molecular species compositions at 15 d after the addition of each FA resembled those of cells with no added FAs. Moreover, some common molecular species in all experimental cells increased, as well as cells with no added FAs. It was concluded that the addition of FAs increases the contents of specific molecular species, but does not affect the synthetic processes of individual TG molecular species.     

Measuring the flow of molecules in cells

No methods proposed thus far have the capability to measure molecular flow in live cells at the single molecule level. Here, we review the potentiality of a newly established method based on the spatial correlation of fluorescence fluctuations at a pair of points in the sample (pair correlation method). The pair correlation function (pCF) offers a unique tool to probe the directionality of intracellular traffic, by measuring the accessibility of the cellular landscape and its role in determining the diffusive routes adopted by molecules. The sensitivity of the pCF method toward detection of barriers means that different structural elements of the cell can be tested in terms of penetrability and mechanisms of regulation imparted on molecular flow. This has been recently demonstrated in a series of studies looking at molecular transport inside live cells. Here, we will review the theory behind detection of barriers to molecular flow, the rules to interpret pCF data, and relevant applications to intracellular transport.     

High Molecular Weight Protease in Rumen Ciliate Protozoa

Mixed rumen ciliate protozoa (mainly Entodiniinae) from goats have two kinds of protease; one has a pH optimum of 3.0, the other is active at neutral or alkaline pH. The protease active at neutral or alkaline pH was partially purified from the supernatant after centrifugation of sonicated mixed rumen ciliate protozoa. The supernatant was chromatographed on Bio-Gel A-1.5m and a partially purified protease was obtained. This protease had a molecular weight of more than 400,000. When the sonicated protozoa were heated at 55°C for 15min, the active peak from the Bio-Gel A-1.5m column was shifted to a lower molecular weight, 27,000. The high molecular weight protease was strongly activated by high temprature and SDS, and inhibited by E-64 c. The protease degraded many proteins including those found in rumen bacteria. These findings suggest that rumen ciliate protozoa have high molecular weight protease that plays a role in the digestion of feed and bacterial protein.     

Sedimentation equilibrium in macromolecular solutions of arbitrary concentration. I. Self-associating proteins

Relations describing sedimentation equilibrium in solutions of self-associating macromolecules at arbitrary concentration are presented. These relations are obtained by using scaled-particle theory to calculate the thermodynamic activity of each species present at a given radial distance. The results are expected to be valid for solutions of globular proteins under conditions such that interactions between individual solute molecules may be approximated by a hard-particle potential. Sedimentation equilibria in solutions containing either a nonassociating solute or a solute that self-associates according to several different schemes are simulated using the derived relations. The results of these simulations are presented in terms of the dependence of apparent weight-average molecular weight upon solute concentration. Simple empirical relations are presented for estimating the true weight-average molecular weight from the apparent weight-average molecular weight, without reference to any particular self-association scheme. The weight-average molecular weight estimated in this fashion is within a few percent of the true weight-average molecular weight at all experimentally realizable solute concentrations ( < 400 g/L).     

beta-Galactosidases in mouse duodenal mucosa.

beta-Galactosidase activity, in fetal mice, first appears at 16 days of gestation and has a pH optimum of 4. In postnatal development the enzyme activity of cell homogenates tends to show bimodal pH at 4 and 5.6. There are two molecular forms of the enzyme, separable both by molecular-sieve chromatography and electrophoresis. One of the molecular forms of the enzyme is active over a wider range of pH (3.2-6.2) and has half as much activity at 5.6 as it does at 4. This isoenzyme is continuously present in both fetal and postnatal stages. The second isoenzyme first appears at birth, is active over a narrower range of pH (4.6-6.2) and inactive at PH 4. The bimodal pH optima observed in postnatal stages in the cell homogenates, appears to be due to the combined activity of the two molecular forms. In isolated brush border membranes, isoenzyme 2 is the only one present. The other organelles (mitochondria, microsomes, lysosomes, nuclei and cytoplasm) have variable proportions of both isoenzymes, as indicated by the activity ratio at pH 4/5.6.     

Denaturation of proteins in methanol/water mixtures

The solvophobic theory developed earlier by Sinanoglu introducing the use of molecular surface areas and microthermodynamic surface and interfacial tensions at molecular dimensions is applied to the interpretation of calorimetric data on denaturation of lysozyme in a wide range of methanol/water mixtures. The experimental values of standard unitary free energies of denaturation correlate well with our predictions. The molecular surface area change of the protein upon denaturation is evaluated using the solvophobic theory. The maximum in the stability of the native form of the protein is predicted to occur at 8% (v/v) methanol. This is found to be in agreement with the experimental results.     

A few low-frequency normal modes predominantly contribute to conformational responses of hen egg white lysozyme in the tetragonal crystal to variations of molecular packing controlled by environmental humidity

The structures of proteins in crystals are fixed by molecular interactions with neighboring molecules, except in non-contacting flexible regions. Thus, it is difficult to imagine what conformational changes occur in solution. However, if molecular interactions can be changed by manipulating molecular packing in crystals, it may be possible to visualize conformational responses of proteins at atomic resolution by diffraction experiments. For this purpose, it is suitable to control the molecular packing in protein crystals by changing the volume of solvent channels through variation of the environmental relative humidity. Here, we studied conformational responses of hen egg white lysozyme (HEWL) in the tetragonal crystal by X-ray diffraction experiments using a humidity-control apparatus, which provided air flow of 20-98%rh at 298 K. First, we monitored the lattice parameters and crystalline order during dehydration and rehydration of HEWL crystal between 61 and 94%rh at 300 K. Then two crystal structures at a resolution of 2.1 ? using diffraction data obtained at 84.2 and 71.9%rh were determined to discuss the conformational responses of HEWL against the external perturbation induced by changes in molecular packing. The structure at 71.9%rh displayed a closure movement that was likely induced by the molecular contacts formed during dehydration and could be approximated by ten low-frequency normal modes for the crystal structure obtained at 84.2%rh. In addition, we observed reorganization of hydration structures at the molecular interfaces between symmetry neighbors. These findings suggest that humidity-controlled X-ray crystallography is an effective tool to investigate the responses of inherent intramolecular motions of proteins to external perturbations.     

The influence of humic substances on the molecular weight distributions of phosphate and iron in epilimnetic lake waters

1. The influence of humic substances on the association of free inorganic iron and phosphate with material of larger molecular weight was investigated in epilimnetic samples from two humus‐rich lakes of contrasting ionic strength. After modification of the molecular weight distribution of the humic substances in the samples using dialysis and ultrafiltration, the molecular weight distribution of added radioisotopes ( Fe3+ and PO43?) was assessed using gel filtration chromatography.
2. The association of Fe3+ and PO43? with larger molecular weight fractions (>50 000 and 10 000–50 000 Da) was not in general related to the quantity of humic substances of the same molecular weight in the samples. However, the proportions of Fe3+ and PO43? observed in higher molecular weight peaks were strongly correlated to the quantity of humic substances of the same molecular weight in (a) the 10 000–50 000 Da peak in the sample of low ionic strength at pH 5.5 and pH 7.0, and (b) the> 50 000 Da peak in the sample of higher ionic strength at pH 4.0.
3. It was concluded that humic substances promote the association of Fe3+ and PO43? with higher molecular weight fractions primarily by acting as peptizing agents for inorganic colloids containing Fe and P. Association of Fe3+ and PO43? with material of higher molecular weight via the formation of humic substance‐Fe3+–PO43? complexes was identified but only at specific pH and within specific molecular weight ranges for each of the epilimnetic lake water samples studied.     

Understanding Actin Organization in Cell Structure through Lattice Based Monte Carlo Simulations

Understanding the connection between mechanics and cell structure requires the exploration of the key molecular constituents responsible for cell shape and motility. One of these molecular bridges is the cytoskeleton, which is involved with intracellular organization and mechanotransduction. In order to examine the structure in cells, we have developed a computational technique that is able to probe the self-assembly of actin filaments through a lattice based Monte Carlo method. We have modeled the polymerization of these filaments based upon the interactions of globular actin through a probabilistic model encompassing both inert and active proteins. The results show similar response to classic ordinary differential equations at low molecular concentrations, but a bi-phasic divergence at realistic concentrations for living mammalian cells. Further, by introducing localized mobility parameters, we are able to simulate molecular gradients that are observed in non-homogeneous protein distributionsin vivo. The method and results have potential applications in cell and molecular biology as well as self-assembly for organic and inorganic systems.     

Molecular feature of the nerve growth factor in human serum

High molecular weight binding components which bind [¹²⁵I] mouse β nerve growth factor exist in human serum. The binding of β nerve growth factor to the serum components was inhibited at alkaline condition. After gel filtration of human serum on a Sephadex G-150 column at neutral condition, the nerve growth factor-like immunoreactivity was observed in only one peak, differing from the high molecular weight serum components. However, at alkaline condition two peaks with nerve growth factor-like immunoreactivity appeared; one was almost at the position observed at neutral pH, and the other was a new peak eluted approximately to the column volume. these results suggest that there are at least two nerve growth factor-like molecules in human serum and most of the nerve growth factor in the serum exists in a complex form associated with serum components with high molecular weight.     

Heterogeneous genomic molecular clocks in primates

Using data from primates, we show that molecular clocks in sites that have been part of a CpG dinucleotide in recent past (CpG sites) and non-CpG sites are of markedly different nature, reflecting differences in their molecular origins. Notably, single nucleotide substitutions at non-CpG sites show clear generation-time dependency, indicating that most of these substitutions occur by errors during DNA replication. On the other hand, substitutions at CpG sites occur relatively constantly over time, as expected from their primary origin due to methylation. Therefore, molecular clocks are heterogeneous even within a genome. Furthermore, we propose that varying frequencies of CpG dinucleotides in different genomic regions may have contributed significantly to conflicting earlier results on rate constancy of mammalian molecular clock. Our conclusion that different regions of genomes follow different molecular clocks should be considered when inferring divergence times using molecular data and in phylogenetic analysis.