Purification and characterization of a common soil component which inhibits the polymerase chain reaction

DNA prepared from soil usually contains a brown-tinted inhibitor of the polymerase chain reaction (PCR) which limits the sensitivity of this technique for specific detection of microorganisms. To localize the inhibitor, soil fractions were tested for their inhibitory effect on the PCR reaction. A highly inhibitory activity, sufficient to account for the inhibition typically exhibited by soil DNA, was found to be tightly associated with the soil microorganism fraction. After cell breakage, the inhibitory material became soluble, and was not separable from DNA by standard purification procedures. A method was derived by which most of the inhibitory material could be selectively solubilized from the microorganism fraction without cell breakage, using successive washes with buffers differing in EDTA concentration. This technique was used to isolate a substance with characteristics suggesting that it is the major PCR inhibitor contaminating DNA purified from soil. It was found to be an organic, water-soluble compound of high molecular weight, and was present in a variety of soil types from different locations. It was found to be distinctly different in its solubility properties from humic and fulvic acids, and also in its FT-IR and NMR spectra. It forms a complex with protein and may inhibit the PCR reaction by an interaction with Taq DNA polymerase.     

Identification of Myosin on the Surface of Wheat Mitochondria

It was demonstrated that myosin was associated with the surface of mitochondria in wheat ( Triticum aestivum L. ). Assays of polyacrylamide gel electrophoresis and Western blotting have shown that a polypeptide with molecular weight of 210 kD could be recognized by a polyclonal antibody against hmnan muscle myosin. It was found that the ATPase activity of mitochondrial suspension could be stimulated by F-actin isolated from chicken muscle, which indicated that there was myosin on the surface of wheat mitochondria. This result was confirmed by electron microscopic observation: mitochondria treated with N-ethylmaleimide (NEM) could be wrapped by the F-actin.     

Purification and characterization of acidolysin, an acidic metalloprotease produced by Clostridium acetobutylicum ATCC 824.

Acidolysin an extracellular protease produced by Clostridium acetobutylicum ATCC 824 was purified to homogeneity by anion-exchange chromatography with a recovery of 91%. The enzyme was a monomeric protein with a molecular weight of 44,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an acidic isoelectric point of 3.3. Acidolysin was very sensitive to metal-chelating agents and phosphoramidon and was unaffected by sulfhydryl reagents. It was shown to be a calcium- and zinc-containing protease. It exhibited optimal activity against Azocoll at pH 5 and 45 degrees C. It was stable at low pH and heat labile above 50 degrees C. It exhibited specificity toward peptide bonds formed by the amino group of hydrophobic amino acids (isoleucine, leucine, and phenylalanine) and its NH2-terminal amino acid sequence showed a high degree of similarity with that of Bacillus subtilis neutral metalloprotease A. Acidolysin is the first phosphoramidon-sensitive, acidic zinc metalloprotease reported.     

Purification and characterization of acidolysin, an acidic metalloprotease produced by Clostridium acetobutylicum ATCC 824

Acidolysin an extracellular protease produced by Clostridium acetobutylicum ATCC 824 was purified to homogeneity by anion-exchange chromatography with a recovery of 91%. The enzyme was a monomeric protein with a molecular weight of 44,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an acidic isoelectric point of 3.3. Acidolysin was very sensitive to metal-chelating agents and phosphoramidon and was unaffected by sulfhydryl reagents. It was shown to be a calcium- and zinc-containing protease. It exhibited optimal activity against Azocoll at pH 5 and 45 degrees C. It was stable at low pH and heat labile above 50 degrees C. It exhibited specificity toward peptide bonds formed by the amino group of hydrophobic amino acids (isoleucine, leucine, and phenylalanine) and its NH2-terminal amino acid sequence showed a high degree of similarity with that of Bacillus subtilis neutral metalloprotease A. Acidolysin is the first phosphoramidon-sensitive, acidic zinc metalloprotease reported.     

The effect of bacteriophage induction on the repair ability ofEscherichia coli 15 TAU

The growth, lysis and survival ofEscherichia coli 15 TAU following UV irradiation was examined. An attempt was made to separate the repair processes and prophage induction by preincubation with chloramphenicol and subsequent incubation with chloramphenicol or caffeine. It was found that the prophage induction does not diminish the repair ability of cells that have survived the induction. It cannot be ascertained whether the cell fraction was induced at all or whether the induction was partially reverted subsequently. It can be concluded that the results derived from examining the survival of tially reverted subsequently. It can be concluded that the results derived from examining the survival of UV-irradiation cultures ofEscherichia coli 15 TAI do not require any correction for phage induction.     

Puff induction in larval salivary gland chromosomes ofDrosophila hydei sturtevant

The puffing pattern in salivary chromosomes of third instar larvae ofDrosophila hydei was studied following treatment with various gases, potassium cyanide, or vacuum. It was found that a number of specific puffs appear when anaerobiosis is followed by exposure to air or oxygen. These puffs seem to be independent of larval age, and are identical with some of those puffs which can be induced by raised temperature. It is suggested that the chromosomal loci involved, are connected with respiration.     

Systemic spread of Campylobacter jejuni after intravenous infections

Mice were infected intravenously with Campylobacter jejuni in order to study systemic translocation of this vibrio, as well as the interactions between bacteria and the host's defense mechanisms. It was found that granulocytes phagocyte C. jejuni in the bloodstream and that phagocytosis could be stimulated with LPS-pretreatment or, less effectively, opsonizing antibodies. It could also be demonstrated that these circulating 'infected' granulocytes are eliminated from the bloodstream mostly by the hepatic Kupffer's cells and that virulent strains of C. jejuni persist in the liver up to thirty days. It has to be concluded that phagocytosis by granulocytes and clearance of C. jejuni from the bloodstream by the liver represent important defense mechanisms in systemic Campylobacter infections.     

An Empirical Trial Of The Effectiveness of Statistical Methodology With Site Survey Data: An Application of The Correlation Coefficient to Survey Data From Mahaska County,Iowa*

Abstract

To find out whether complex statistical methods could be successfully applied to site survey data, the correlation coefficient was used to evaluate the reliability and validity of survey data gathered from Mahaska county, Iowa by means of a mailed questionnaire. It was also used to identify geographical characteristics of the sites located by the above survey. The reliability of the survey data was found to be questionable, while their validity was found to be good. Sites in Mahaska county were found to be located near rivers, at low altitudes, and on timber soil. It was concluded that the correlation coefficient can be a useful tool in working with site survey data.     

An autoclavable electronic interface for germfree isolators

An electronic interface was designed and fabricated for use with germfree isolators. It allows a variety of sensors or electrical instruments inside an isolator to be readily connected to monitoring or activating devices on the outside of an isolator. It was built into a 25 cm diameter cylinder whose open ends were loosely covered by an inner and outer connector panel. These two panels were wired together through an airtight dividing wall placed transversely across the middle of the cylinder. The interface was mounted in an entry port of a stainless steel isolator and steam sterilized with the isolator. It has proven to be a useful, contamination-free, durable device.     

Antiviral Agent from λ-infected Escherichia coli K-12: I. Isolation

Phagicin, which is an antiviral agent active against deoxyribonucleic acid (DNA) viruses such as vaccinia and herpes simplex, has been identified as a phage internal protein. It was found in infected Escherichia coli lysates, but could also be obtained by disruption of the purified infective particles after incubation with LiCl at 46 C for 15 min or by sonic treatment. After centrifugation at high speed, the antiviral activity was found in the DNA phase and could be separated by chromatography on Sephadex gels with 0.2 M phosphate buffer (pH 7.5) as the eluent. Phagicin present in lysates after removal of infective particles was nondialyzable and was bound to nucleic acids. It could be released during precipitation of nucleic acids by streptomycin sulfate, and in this form it could be easily dialyzed. The antiviral activity of phagicin was specific for herpes simplex and vaccinia viruses.     

Tumor-degenerating factor (TDF)]

A new human fibroblast-producing factor which degenerates human tumor cells in vitro was found and designated as tumor-degenerating factor (TDF). The characteristics of the TDF were as follows: 1) The molecular weight of the TDF was estimated to be 30k by SDS-PAGE. 2) It may be basic and glycosylated. 3) Its activity was inhibited by fibronectin and, vice versa, by binding TDF molecule with fibronectin molecule. 4) Its activity was enhanced by interferons. It is possible that the TDF is an important substance concerning the interaction between tumor cells and interstitial cells.     

Systemic spread of Campylobacter jejuni after intravenous infections

Abstract Mice were infected intravenously with Campylobacter jejuni in order to study systemic translocation of this vibrio, as well as the interactions between bacteria and the host's defense mechanisms. It was found that granulocytes phagocyte C. jejuni in the bloodstream and that phagocytosis could be stimulated with LPS-pretreatment or, less effectively, opsonizing antibodies. It could also be demonstrated that these circulating 'infected' granulocytes are eliminated from the bloodstream mostly by the hepatic Kupffer's cells and that virulent strains of C. jejuni persist in the liver up to thirty days. It has to be concluded that phagocytosis by granulocytes and clearance of C. jejuni from the bloodstream by the liver represent important defense mechanisms in systemic Campylobacter infections.     

The Effect of Light on the Translocation from Sugar-beet Leaves

Illumination was found to increase the rate of translocationfrom sugar-beet leaves. This stimulation of translocation wasnot dependent on the concentration of either sugar or ATP inthe leaf. It was, however, dependent on carbon fixation by theleaf and it increased with light intensity in a manner similarto that of assimilation rate. It was evident that these parallelstimulations by light might have a common cause, concerningthe nature of which some suggestions are made. It was also shownthat under normal daylight conditions other factors affectedtranslocation rate and that by suitable experiments their effectson translocation could be determined and shown to act independently.     

Cytodiagnosis of hepatic amyloidosis by fine needle aspiration cytology: a case report

BACKGROUND: Amyloid in fine needle aspirates tends to be overlooked due to its rarity. It may appear at virtually any site and can be identified by special stains. CASE: In a case of amyloidosis of the liver, where fine needle aspiration cytology was instrumental in making the primary diagnosis. Smears revealed normal hepatocytes and chronic inflammatory cells. Amorphous material (amyloid) was extensively present between compressed hepatocytic clusters. It stained pale green in Papanicolaou-stained preparations and reddish purple with Giemsa stain. This material was initially thought to be hyalinized granulomas. However, it was later confirmed to be amyloid by using Congo Red stain. CONCLUSION: Whenever amorphous material is present in smears, a high index of suspicion for amyloid needs to be maintained.     

Purification and properties of cytochrome b556 in the respiratory chain of aerobically grown Escherichia coli K12.

Cytochrome b556, a major component of type b cytochromes in the respiratory chain of aerobically grown Escherichia coli, was purified to near homogeneity. It was solubilized from cytoplasmic membranes by treatment with Sarkosyl/cholate mixture and purified by gel filtration on Sephadex G-200. The purified cytochrome b556 is an oligomer composed of identical polypeptides, with a molecular weight of 17,500, determined by gel electrophoresis in the presence of sodium dodecyl sulfate. It contains equimolar amounts of heme and polypeptide but no detectable non-heme iron, phospholipid, or dehydrogenase. Its isoelectric point was determined to be 8.5. The cytochrome b556 is highly hydrophobic in its amino acid composition and does not contain any half-cystine residues. The purified cytochrome b556 is spectrophotometrically pure and the alpha absorption peak in its difference spectrum at 77 K is at 556 nm. The molar extinction coefficient of cytochrome b556 was determined as 22.8 cm-1 mM-1. Its oxidation-reduction potential was found to be -45 mV. It could be reduced by D-lactate dehydrogenase of E. coli in the presence of menadione.     

Study of a yeast mutant with modified mitochondrial translocation of adenine nucleotides

The effect of ethidium bromide on the growth of a yeast mutant with an impaired mitochrondrial translocation system of adenine nucleotides (op-1 mutant) was investigated. It was found that the op-1 mutant stops growing both under growing and non-growing conditions after treatment with ethidium bromide and that the growth cannot be restored by adding low-molecular compounds to the growth medium. It was the aim of the experiments to clarify whether the cessation of growth of the op-1 mutant after induction of the rho- mutation can be simulated by inhibitors phenotypically changing the mitochondrial function. It appears likely that the op-1 mutant stops growing only after the rho- mutation has been induced, because the phenotypic simulation of the rho- mutation does not lead the cessation of growth of the op-1 mutant.     

Zearalenone-Like Substance in Winter Plants and Its Relation to Vernalization

It was found that there is a zearalenone-like substance in the growing points of vernalized wheat seedlings and carrot plants. The content of this substance synchronously increased with the depth of vernalization. It was separated by thin layer chromatography. Its UV spectrum was similar to that of zearalenone. It is believed that this substance may be one of the important endogenous substances which control the vernalization of winter plants.     

Catalase activity as affected by carnosine and sodium nitrite in vitro

It was shown in vitro that the activity of catalase Penicillium vitale decreases when treated by sodium nitrite to the greater extent than when affected by dipeptide carnosine (beta-alanyl-L-histidine). At alternating introduction of carnosine and NaNO2 that component affected the catalase activity which was the first to be introduced. The entering of the next metabolite into the medium did not change the enzyme activity. It is shown that carnosine binds with a molecule of catalase. Carnosine may be considered one of natural regulators of catalase activity.     

The effect of neuraminidase treatment of ribosome-free membranes on their ribosomal reattachment ability

Ribosome-free membranes prepared by means of 2M lithium chloride lose 90% of their ability to reattach ribosomes after digestion with neuraminidase for 1 hour. It can be shown that this is not due to proteolytic degradation of the membranes by contaminating proteases presant in neuraminidase preparations. It was found that neuraminidase troatment of rat liver RER results in the release of approximately 50% of the bound ribosomes. Ribosomal reattachment ability can be restored to the neuraminidase-treated membranes by incubation with N-acetylneuraminic acid and nucleotide triphosphates.     

A halophilic serine proteinase from Halobacillus sp. SR5-3 isolated from fish sauce: purification and characterization

A halophilic bacterium was isolated from fish sauce, classified, and named Halobacillus sp. SR5-3. A purified 43-kDa proteinase produced by this bacterium showed optimal activity at 50 degrees C and pH 9-10 in 20% NaCl. The activity of the enzyme was enhanced about 2.5-fold by the addition of 20-35% NaCl, and the enzyme was highly stabilized by NaCl. It was found to be a serine proteinase related to either chymotrypsin or subtilisin. It absolutely preferred Ile at the P(2) position of substrates. Thus, the enzyme was found to be a halophilic serine proteinase with unique substrate specificity.