EVALUATION OF BEADRETRIEVERTM, AN AUTOMATED SYSTEM FOR CONCENTRATION OF ESCHERICHIA COLI O157 FROM ENRICHMENT CULTURES BY IMMUNOMAGNETIC SEPARATION

The aim of this study was to compare automated immunomagnetic separation (AIMS) with manual IMS (MIMS) for detection of E. coli O157 in natural and artificially inoculated samples. E. coli O157 was isolated from 27 (14.7%) of 184 natural bovine fecal samples by MIMS and from 26 (14.1%) by AIMS. Of 94 natural carcass samples, E. coli O157 was isolated from 6 by both MIMS and AIMS and from a further 6 by AIMS only. Three of the six positive only by AIMS were from tubes processed adjacently to one containing an inoculated sample and strains isolated from these adjacent tubes were indistinguishable by various typing techniques. Of the 36 carcass samples inoculated with E. coli O157, the organism was isolated from 25 by both MIMS and AIMS and from a further 9 by AIMS only. To process 100 samples by MIMS took 5h whereas by AIMS this took only 3h 20 min which represented a considerable saving of staff time.     

Rapid isolation of plant peroxidase. Purification of peroxidase a from Petunia

A rapid isolation procedure was developed for purification of peroxidase a from Petunia hybrida . Rapid isolation was possible since about 15% of the extracellular protein from stem tissue obtained by vacuum infiltration followed by centrifugation of the tissue represents peroxidase. Purification of peroxidase a from intercellular fluid was achieved by two acetone precipitation steps followed by DEAE-cellulose chromatography.
Three different forms of peroxidase were eluted from DEAE-cellulose at different NaCl concentrations. Isoelectric focusing showed, however, a pI of 3.8 for all three forms of peroxidase a . Only part of the peroxidase a enzymes bound to Concanavalin A indicating heterogeneity in the carbohydrate part. Homology of peroxidase a to the peroxidase G₁ group from tobacco is discussed.     

Multiple structural genes for mouse amylase

Salivary and pancreatic amylases from the mouse show both structural and quantitative genetic variation encoded within a gene complex on chromosome 3. Two fundamental questions prompted by this variation are whether salivary and pancreatic amylases are derived from different structural genes and whether multiple structural genes are causing the quantitative variation observed in each of the two amylases. These questions were approached by comparing the amylase protein from 12 congenic lines carrying amylase gene complexes derived from different origins. The amylases were purified by affinity chromatography employing the inhibitor cyclohepta-amylose and characterized in terms of amino acid composition, specific activity, molecular weight, and heat stability. They were analyzed by native electrophoresis in polyacrylamide gels and by peptide mapping employing both cyanogen bromide cleavage and restricted proteolysis in the presence of dodecylsulfate. By these techniques, many differences in the structure of pancreatic amylase that were not reflected in the salivary amylase were found among mouse strains. Likewise, a distinct salivary amylase variant was found. These results suggest that independent structural genes exist for the two amylases. Furthermore, by all criteria used, pancreatic amylase from single strains exhibits molecular heterogeneity, whereas heterogeneity was never found for salivary amylase. We conclude that at least four structural genes code for pancreatic amylase while only a single gene, different from any of the pancreatic genes, codes for salivary amylase.This work was supported by grants from the Danish Natural Science Research Council and a grant from the United States Public Health Service (Grant GM-19521). Part of the study was made during a 1-month visit of A. J. L. in Aarhus, which was supported by grants from NATO and the University of Aarhus.     

Altered purine and pyrimidine metabolism in erythrocytes with purine nucleoside phosphorylase deficiency

Purine and pyrimidine metabolism was compared in erythrocytes from three patients from two families with purine nucleoside phosphorylase deficiency and T-cell immunodeficiency, one heterozygote subject for this enzyme deficiency, one patient with a complete deficiency of hypoxanthine-guanine phosphoribosyltransferase, and two normal subjects. The erythrocytes from the heterozygote subject were indistinguishable from the normal erythrocytes. The purine nucleoside phosphorylase deficient erythrocytes had a block in the conversion of inosine to hypoxanthine. The erythrocytes with 0.07% of normal purine nucleoside phosphorylase activity resembled erythrocytes with hypoxanthine-guanine phosphoribosyltransferase deficiency by having an elevated intracellular concentration of PP-ribose-P, increased synthesis of PP-ribose-P, and an elevated rate of carbon dioxide release from orotic acid during its conversion to UMP. Two hypotheses to account for the associated immunodeficiency—that the enzyme deficiency leads to a block of PP-ribose-P synthesis or inhibition of pyrimidine synthesis—could not be supported by observations in erythrocytes from both enzyme-deficient families.This work was supported by U.S. Public Health Service Grant AM 19674 and 5 M01 RR 42 and by a Grant-In-Aid from American Heart Association (77-849) and with funds contributed in part by the Michigan Heart Association. N.L.E. is a Rheumatology Fellow from the Rackman Arthritis Research Unit supported by Training Grant USPHS AM 07080.     

Peroxisome-desmosome complexes in mouse hepatic cells

Summary In addition to mitochondrion-desmosome complexes, peroxisome-desmosome complexes were present in mouse hepatocytes. The latter complexes consisted of a desmosome which was flanked on one or both sides by a peroxisome. Occasional desmosomes were confronted on one side by a peroxisome and on the other by a mitochondrion. It is suggested that the association between organelles and desmosomes is fortuitous, and that no functional significance can be inferred from this association.This work was supported in part by grants from the Heart Association of Northeastern Ohio, Inc., by grant 3C179 from the Cleveland Foundation, by American Cancer Society Institutional Grant In-57-H, and by grant 5 SO1 FR05335-09 from the National Institutes of Health. The expert technical assistance of Merry A. Hetrick and Jeanne Luschin is acknowledged.     

Identification of the secreted macromolecular immunogens of Staphylococcus aureus by analysis of serum

The ability of sera to recognise secreted macromolecules of Staphylococcus aureus was examined by ELISA and Western immunoblotting. Individual secreted proteins were also studied using both human sera and sera from rabbits immunised with secreted macromolecules. Patients sera showed a wide range of IgG antibody titres to secreted macromolecules and whole bacteria. Controls showed a significantly lower IgG response. Western immunoblotting revealed that a significant number of secreted proteins were recognised by circulating IgG antibodies. Surprisingly, both the sera from controls and from patients recognised similar macromolecules including a number of potential virulence factors. The major difference was in the IgG binding to a 16-kDa component, which was recognised by the majority of the sera from infected individuals, but only by a small number of sera from healthy controls. The higher incidence of antibodies recognising the 16 kDa component may be related to our earlier finding that the major bone resorbing component of S. aureus is a heterodimeric protein containing a 16-kDa subunit, the activity of which could be blocked by sera.     

A method for the isolation and serial propagation of keratinocytes,endothelial cells,and fibroblasts from a single punch biopsy of human skin

Summary When multiple types of cells from normal and diseased human skin are required, techniques to isolate cells from small skin biopsies would facilitate experimental studies. The purpose of this investigation was to develop a method for the isolation and propagation of three major cell types (keratinocytes, microvascular endothelial cells, and fibroblasts) from a 4-mm punch biopsy of human skin. To isolate and propagate keratinocytes from a punch biopsy, the epidermis was separated from the dermis by treatment with dispase. Keratinocytes were dissociated from the epidermis by trypsin and plated on a collagen-coated tissue culture petri dish. A combination of two commercial media (Serum-Free Medium and Medium 154) provided optimal growth conditions. To isolate and propagate microvascular endothelial cells from the dermis, cells were released following dispase incubation and plated on a gelatin-coated tissue culture dish. Supplementation of a standard growth medium with a medium conditioned by mouse 3T3 cells was required for the establishment and growth of these cells. Epithelioid endothelial cells were separated from spindle-shaped endothelial cells and from dendritic cells by selective attachment toUlex europeus agglutinin I-coated paramagnetic beads. To establish fibroblasts, dermal explants depleted of keratinocytes and endothelial cells were attached to plastic by centrifugation, and fibroblasts were obtained by explant culture and grown in Dulbecco’s modified Eagle’s medium (DMEM) containing fetal bovine serum (FBS). Using these isolation methods and growth conditions, two confluent T-75 flasks of keratinocytes, one confluent T-25 flask of purified endothelial cells, and one confluent T-25 flask of fibroblasts could be routinely obtained from a 4-mm punch biopsy of human skin. This method should prove useful in studies of human skin where three cell types must be grown in sufficient quantities for molecular and biochemical analysis.     

Predators can induce swarming behaviour and locomotory responses in Daphnia

1. Exposure to chemical cues released by fish induced a tendency to form and maintain aggregations in two Daphnia clones originating from habitats inhabited by fish (and invertebrate predators), while Daphnia from a clone originating from a fishless habitat did not aggregate in response to fish cues.
2. Daphnia from one of the two clones responsive to fish cues, also aggregated in response to chemical cues released by invertebrate predators or to a substance released from homogenized Daphnia .
3. Chemical cues released by predators and the substance released from homogenized conspecifics induced a significant decrease in Daphnia' s swimming speed and an increased readiness to perform a somersaulting escape behaviour, especially in response to light cues.     

Characterization of the Tumorlike (T) Antigen Induced by Type 12 Adenovirus : I. Purification of the Antigen from Infected KB Cells and a Hamster Tumor Cell Line

The T antigen induced by type 12 adenovirus was purified from KB cells infected in the presence of 10(-6)m 5-fluoro-2-deoxyuridine to inhibit synthesis of viral capsid antigens. The antigen was purified approximately 200-fold, and the purified product contained only negligible amounts of host-cell contaminants, as judged by the residual radioactivity from (14)C-labeled uninfected cells which had been added to infected cells at the initiation of the purification. Immunoelectrophoresis indicated that the purified T-antigen preparation contained a single antigenic species. The T antigen from a hamster cell line (HT-1) derived from a type 12 adenovirus-induced tumor was purified by the same procedure. The T antigens from the two different sources were shown to be immunologically similar by use of a rabbit antiserum prepared against the purified T antigen from infected KB cells and sera from hamsters bearing tumors induced by type 12 adenovirus.     

黑曲霉CGMCC0774和朱红密孔菌CGMCC1115两步转化阿魏酸制备生物香兰素

在25 L发酵罐中黑曲霉Aspergillus niger CGMCC0774转化阿魏酸可生成香草酸2.24 g/L,摩尔转化率64.6%;朱红密孔菌Pycnoporus cinnabarinus CGMCC1115转化提取的香草酸可生成香草醛1.45 g/L,摩尔转化率为79.9%。将两步微生物转化有机串联,即用黑曲霉转化液加预先培养的朱红密孔菌Pycnoporus cinnabarinus CGMCC1115菌丝体继续转化,可产香草醛1.06 g/L,对原料阿魏酸的摩尔转化率34.0%。用米糠提取的天然阿魏酸做原料,两步串联微生物转化制备的生物香兰素经13C同位素的分析,符合生物香草素的等同要求。     

Inhibition of protein synthesis in vitro by a lectin from Momordica charantia and by other haemagglutinins.

Protein synthesis by a rabbit reticulocyte lysate is inhibited by the haemagglutinating lectins from Momordica charantia and Crotalaria juncea seeds and from the roe of Rutilus rutilus, and by a commercial preparation of the mitogenic lectin from Phytolacca americana. The haemagglutinins from the seeds of Ricinus communis and of Vicia cracca acquired inhibitory activity after their reduction with 2-mercaptoethanol.     

One-step purification of metallothionein extracted from two different sources

We describe a one-step purification of hepatic metallothionein from the Amazon fish Colossoma macropomum injected with cadmium and from the copper-loaded metallothionein from the yeast Saccharomyces cerevisiae, performed by affinity chromatography through metal-chelating columns. Yeast metallothionein was purified from Cu2+-loaded resin and eluted by a continuous EDTA gradient whereas hepatic metallothionein extracted from fishes was purified by Ni2+-loaded resin and eluted by a continuous imidazol gradient. Purified metallothioneins were evaluated by SDS-PAGE and characterized by UV spectra of the apo- and Cd2+-loaded protein. This method allowed high purity and yield as well as rapid one-step extraction of both metal-loaded and apoprotein.     

一种新型淀粉酶的鉴定及其产酶菌株的筛选

对筛选到的菌株ZX99产生的一种新型淀粉酶（异麦芽低聚糖酶）进行了分析鉴定,ZX99菌株能产生一种胞外淀粉酶,该酶能催化淀粉的降解产生异麦芽低聚糖,对原产酶菌株ZX99多次进行紫外线照射诱变后,获得了优良,稳定的变异菌株RB3．232,其产酶水平为原株的160％,产物薄层层析证明,该酶能催化淀粉的降解,产生异麦芽糖,潘糖,异麦芽三糖和异麦芽四糖等低聚糖,但对普鲁兰基本不起作用,由此证明它是一种不同于新型普鲁兰酶（nepullulanase)和 传统淀粉酶（amylase)的一种新型淀粉酶。     

Reconstitution of binding protein dependent ribose transport in spheroplasts derived from a binding protein negative escherichia coli K12 mutant and from salmonella typhimurium

Highly purified ribose-binding protein from Escherichia coli has been used to reconstitute a binding-protein-dependent ribose transport in spheroplasts derived from a binding-protein-deficient mutant of E coli K 12, and in spheroplasts derived from Salmonella typhimurium. The cross-species reconstitution was nearly as efficient as the reconstitution of the E coli strain from which the binding protein was derived. Antibody raised against the ribose binding protein completely prevented reconstitution, whereas it had no effect on whole cells. The reconstitution procedure has been improved by generating spheroplasts from cells grown in a rich medium and by reducing the background uptake in spheroplasts through a special washing procedure. Rapid purification of ribose binding protein by high pressure liquid chromatography is also described.     

The capsular turnover product of Staphylococcus aureus strain Smith

Abstract The capsular polysaccharide released from the bacterial surface by cell wall turnover during growth exhibited less size heterogeneity and a higher average molecular mass than the polysaccharide extracted from the cell by treatment with lysostaphin or low pH. Treatment of turnover polysaccharide, radiolabelled by growth of the bacteria in the presence of N-acetyl-[³H]-glucosamine, with muramidase B from Chalaropsis released a low molecular weight product chromatographically identical to the peptidoglycan degradation products released from the peptidoglycan-teichoic acid complex by the same treatment. It is concluded that some or all of the capsular polysaccharide released into the culture fluid during growth is derived from peptidoglycan-linked capsular material, solubilised by cell wall turnover.     

Synthetic applications of enantioselective protonation and case study for (S)-alpha-damascone

Among the fragrance compounds synthesized by enantioselective protonation, (S)-alpha-damascone, (R)-muscone, and (S,S)-Vulcanolide are the most prominent ones. (S)-alpha-damascone has been prepared by four different procedures: from the magnesium enolate, from the lithium enolate, from the enol, and from the corresponding thiol ester. We now present a new, industrially viable protocol for the addition of allyl magnesium chloride to the 'cyclogeranoketene' by a Barbier reaction, followed by protonation of the ensuing magnesium enolate by an aggregate formed from (-)-N-isopropylephedrine, lithium isopropylate, and acetic acid, furnishing (S)-alpha-damascone in 91% yield and with 71% ee.     

Chemiosmotic lysis and insulin secretion: studies of isolated granules, intact and permeabilised rat islets of Langerhans

The possible involvement of chemiosmotic lysis of secretory granules in the exocytosis of insulin from pancreatic beta cells was investigated by comparing insulin release from isolated secretory granules, from intact islets of Langerhans, and from electrically permeabilised islets. Lysis of isolated granules was stimulated by ATP in the presence of Mg2+. ATP-induced granule lysis was pH and temperature dependent and was inhibited by collapsing the pH gradient across the granule membrane by removal of permeant anions, or by increasing the extragranular osmolarity. However, insulin secretion from intact islets in response to glucose, a phosphodiesterase inhibitor or a Ca2+ ionophore was only partially inhibited by anion replacement, while Ca2+ -induced insulin release from electrically permeabilised islets was not affected by altering the extragranular or intragranular pH. These results suggest that studies of the stability of isolated granules in vitro do not necessarily relate to insulin release from whole cells, and do not support a major role for chemiosmotic lysis of secretory granules in the exocytotic release of insulin.     

Multiple displacement amplification in combination with high-fidelity PCR improves detection of bacteria from single females or eggs of Metaseiulus occidentalis (Nesbitt) (Acari: Phytoseiidae)

Amplifying microbial DNA by the polymerase chain reaction (PCR) from single phytoseiid mites has been difficult, perhaps due to the low titer of bacteria and to interference by the relatively larger amounts of mite genomic DNA. In this paper we evaluate the efficiency of standard and high-fidelity PCR protocols subsequent to amplification of the whole genome by a multiple displacement amplification (MDA) procedure developed by Dean et al. DNA from the phytoseiid Phytoseiulus persimilis (Athias-Henriot) was tested because it lacks a Cytophaga-like organism (CLO) and we could add known amounts of a plasmid containing a cloned 16S rRNA gene fragment from a CLO from Metaseiulus occidentalis (Nesbitt). P. persimilis genomic DNA was mixed with the serially diluted plasmid and amplified using MDA followed by either standard or high-fidelity PCR. MDA followed by high-fidelity PCR was most efficient and successfully amplified an expected 1.5-kb band from as little as 0.01fg of the plasmid, which is equivalent to about 1 copy. MDA followed by high-fidelity PCR also consistently amplified Wolbachia- or CLO-specific products from naturally infected single females or eggs of M. occidentalis, which will allow detailed studies of infection frequency and transmission of several microorganisms associated with this predatory mite.     

Comparison of the carbon-sequestering abilities of pineapple leaf residue chars produced by controlled combustion and by field burning

This study was undertaken to compare the chemical properties and yields of pineapple leaf residue (PLR) char produced by field burning (CF) with that produced by a partial combustion of air-dried PLR at 340 °C for 3 h in a furnace (CL). Higher total C, lignin content, and yield from CL as well as the presence of aromatic compounds in the Fourier Transform Infrared spectra of the char produced from CL suggest that the CL process was better in sequestering C than was the CF process. Although the C/N ratio of char produced from CL was low indicating a high N content of the char, the C in the char produced from CL was dominated by lignin suggesting that the decomposition of char produced from CL would be slow. To sequester C by char application, the PLR should be combusted in a controlled process rather than by burning in the field.     

Ferulic acid degradation encoded by a catabolic plasmid

Abstract A strain of Pseudomonas fluorescens was isolated from soil with ferulic acid as sole source of carbon and energy. The ability to degrade this compound iis plasmid-encoded, as shown by the transfer of such degradative capacity from the wild-type into a recipient strain by conjugation and isolation of a plasmid from the exconjugants, identical to that from the wild-type and the exconjugants.