The use of poly(L-proline)-Sepharose in the isolation of profilin and profilactin complexes

In the purification of proline hydroxylase by affinity chromatography on poly(L-proline)-Sepharose it was found earlier that two other components, profilin and the complex profilin-actin, also bind with high affinity to this matrix. We have exploited this observation to develop a rapid procedure for the isolation of profilin and profilin-actin complexes in high yields directly from high-speed supernatants of crude tissue-extracts. Through an extensive search for elution conditions, avoiding poly(L-proline) as the desorbant, we have found that active proteins can be recovered from the affinity column with a buffer containing 30% dimethyl sulphoxide. Subsequent chromatography on hydroxylapatite separates free profilin and the two isoforms of profilactin, profilin-actin_β and profilin-actin_γ. The profilin-actin complexes produced this way have high specific activities in the DNAase-inhibition assay, give rise to filaments on addition of Mg²⁺, and can be crystallized. From the isolated profilin-actin complexes the β- and γ-actin isoforms of non-muscle cells can easily be prepared in a polymerization competent form. Pure profilin is either obtained from an excess pool present in some extracts or by dissociation of profilin-actin complexes and removal of the actin.     

Identification and characterization of alkali-tolerant clostridia isolated from biodeteriorated casein-containing building materials

Summary Reports on malodour in buildings constructed in the late 1970s gave rise to thorough investigations on the possible role of vapours of chemical compounds emitted by building materials. The odour could be related to the use of casein as an additive to improve the fluidity of concrete materials used as a self-levelling floor topping compound. Casein was suggested to be degraded by microorganisms, resulting in an accumulation of malodorous substances in the topping compounds.Bacteria isolated from biodeteriorated concrete materials containing caseins exhibited unusual tolerance towards high pH. Two dominant species were found among a total of 80 sporeforming, anaerobic isolates from concrete and raw products of caseins, namely Clostridium bifermentans and Clostridium sporogenes. C. bifermentans had a maximal pH tolerance of 12.2 while C. sporogenes could reproduce up to pH 11.7. The study includes the identification of the clostridia with API multitest as well as an investigation of the volatile organic acid and monoamine patterns. About 100 cfu clostridia/g material could be obtained during the isolation procedures.     

Genetic and blood coagulation characterization of “Swedish” families with von Willebrand's disease types I and III: New aspects of heredity

Summary Twenty-five patients with von Willebrand's disease (vWD) type III were analysed with regard to blood coagulation variables and possible deletions. Nine of the probands and their families were further investigated with DNA linkage analyses. Different patterns of heredity can be suggested in our families with vWD type III, on the basis of blood coagulation analyses. The findings suggest homozygosity in five families and the possibility of compound heterozygosity or a new mutation in the proband in three families. The linkage analyses confirm the results of the coagulation analyses. The segregation of the von Willebrand factor (vWF) gene can be followed in the families, and carrier diagnosis can be made in several of the probands' relatives. The possibility of large deletions in the vWF gene of the probands and their parents was investigated with probes representing the whole vWF cDNA. No deletions were found.This study was approved by the Ethics Committee of the Karolinska Hospital (No. 84:1)     

The 3′→5′ exonuclease associated with HeLa DNA polymerase epsilon

The 3′→5′ exonuclease activity of highly purified large form of human DNA polymerase epsilon was studied. The activity removes mononucleotides from the 3′ end of an oligonucleotide with a non-processive mechanism and leaves 5′-terminal trinucleotide non-hydrolyzed. This is the case both with single-stranded oligonucleotides and with oligonucleotides annealed to complementary regions of M13DNA. However, the reaction rates with single-stranded oligonucleotides are at least ten-fold when compared to those with completely base-paired oligonucleotides. Conceivably, mismatched 3′ end of an oligonucleotide annealed to M13DNA is rapidly removed and the hydrolysis is slown down when double-stranded region is reached. The preferential removal of a non-complementary 3′ end and the non-processive mechanism are consistent with anticipated proofreading function. In addition to the 3′→5′ exonuclease activity, an 5′→3′ exonuclease activity is often present even in relatively highly purified DNA polymerase epsilon preparates suggesting that such an activity may be an essential com-ponent for the action of this enzymein vivo. Contrary to the 3′→5′ exonuclease activity, the 5′→3′ exonuclease is separable from the polymerase activity.     

The 3′→5′ exonuclease associated with HeLa DNA polymerase epsilon

The 3′→5′ exonuclease activity of highly purified large form of human DNA polymerase epsilon was studied. The activity removes mononucleotides from the 3′ end of an oligonucleotide with a non-processive mechanism and leaves 5′-terminal trinucleotide non-hydrolyzed. This is the case both with single-stranded oligonucleotides and with oligonucleotides annealed to complementary regions of M13DNA. However, the reaction rates with single-stranded oligonucleotides are at least ten-fold when compared to those with completely base-paired oligonucleotides. Conceivably, mismatched 3′ end of an oligonucleotide annealed to M13DNA is rapidly removed and the hydrolysis is slown down when double-stranded region is reached. The preferential removal of a non-complementary 3′ end and the non-processive mechanism are consistent with anticipated proofreading function. In addition to the 3′→5′ exonuclease activity, an 5′→3′ exonuclease activity is often present even in relatively highly purified DNA polymerase epsilon preparates suggesting that such an activity may be an essential com-ponent for the action of this enzymein vivo. Contrary to the 3′→5′ exonuclease activity, the 5′→3′ exonuclease is separable from the polymerase activity.     

Large-Scale Production of Nanographite by Tube-Shear Exfoliation in Water

The number of applications based on graphene, few-layer graphene, and nanographite is rapidly increasing. A large-scale process for production of these materials is critically needed to achieve cost-effective commercial products. Here, we present a novel process to mechanically exfoliate industrial quantities of nanographite from graphite in an aqueous environment with low energy consumption and at controlled shear conditions. This process, based on hydrodynamic tube shearing, produced nanometer-thick and micrometer-wide flakes of nanographite with a production rate exceeding 500 gh^-1 with an energy consumption about 10 Whg^-1. In addition, to facilitate large-area coating, we show that the nanographite can be mixed with nanofibrillated cellulose in the process to form highly conductive, robust and environmentally friendly composites. This composite has a sheet resistance below 1.75 Ω/sq and an electrical resistivity of 1.39×10^-4 Ωm and may find use in several applications, from supercapacitors and batteries to printed electronics and solar cells. A batch of 100 liter was processed in less than 4 hours. The design of the process allow scaling to even larger volumes and the low energy consumption indicates a low-cost process.     

Alleged Approach-Avoidance Conflict for Food Stimuli in Binge Eating Disorder

ObjectiveFood stimuli are omnipresent and naturally primary reinforcing stimuli. One explanation for the intake of high amounts of food in binge eating disorder (BED) is a deviant valuation process. Valuation of food stimuli is supposed to influence approach or avoidance behaviour towards food. Focusing on self-reported and indirect (facial electromyography) valuation process, motivational aspects in the processing of food stimuli were investigated.MethodsWe compared an overweight sample with BED (BED+) with an overweight sample without BED (BED-) and with normal weight controls (NWC) regarding their self-reported and indirect (via facial electromyography) valuation of food versus non-food stimuli.ResultsRegarding the self-reported valuation, the BED+ sample showed a significantly stronger food-bias compared to the BED- sample, as food stimuli were rated as significantly more positive than the non-food stimuli in the BED+ sample. This self-reported valuation pattern could not be displayed in the indirect valuation. Food stimuli evoked negative indirect valuation in all groups. The BED+ sample showed the plainest approach-avoidance conflict marked by a diverging self-reported (positive) and indirect (negative) valuation of food stimuli.ConclusionsBED+ showed a deviant self-reported valuation of food as compared to BED-. The valuation process of the BED+ sample seems to be characterized by a motivational ambivalence. This ambivalence should be subject of further studies and may be of potential use for therapeutic interventions.     

Genetic analysis of the polymorphism of the human apolipoprotein E using automated solid-phase sequencing

A direct sequencing approach has been used to analyze the polymorphism in the human apolipoprotein E gene. A method is described, in which the DNA is amplified by the polymerase chain reaction, immobilized, and sequenced by a semi-automatic procedure adaptable to clinical diagnosis. The three alleles of the apolipoprotein E gene, which differ from each other by two nucleotide substitutions and which influence serum cholesterol levels, were analyzed. The solid-phase method was able to resolve the correct nucleotide sequence in samples from both homozygous and heterozygous individuals. No cloning steps are needed and the immobilization and separation of the DNA is accomplished using magnetic beads.     

BRCT domain-containing protein TopBP1 functions in DNA replication and damage response

Topoisomerase IIbeta-binding protein (TopBP1), a human protein with eight BRCT domains, is similar to Saccharomyces cerevisiae Dpb11 and Schizosaccharomyces pombe Cut5 checkpoint proteins and closely related to Drosophila Mus101. We show that human TopBP1 is required for DNA replication and that it interacts with DNA polymerase epsilon. In S phase TopBP1 colocalizes with Brca1 to foci that do not represent sites of ongoing DNA replication. Inhibition of DNA synthesis leads to relocalization of TopBP1 together with Brca1 to replication forks, suggesting a role in rescue of stalled forks. DNA damage induces formation of distinct TopBP1 foci that colocalize with Brca1 in S phase, but not in G(1) phase. We also show that TopBP1 interacts with the checkpoint protein hRad9. Thus, these results implicate TopBP1 in replication and checkpoint functions.