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PubMed1963

INORGANIC POLYPHOSPHATE METABOLISM IN CHLOROBIUM THIOSULFATOPHILUM.

Hughes, D. E. (Dartmouth Medical School, Hanover, N.H.), S. F. Conti, and R. C. Fuller. Inorganic polyphosphate metabolism in Chlorobium thiosulfatophilum. J. Bacteriol. 85:577-584. 1963.-Cells of the obligate phototroph Chlorobium thiosulfatophilum, when grown on a normal concentration of inorganic phosphate, accumulated large intracellular metachromatic granules identified as polymetaphosphate. Inorganic phosphate was released from polymetaphosphate by cell-free extracts. This release was adenosine diphosphate-dependent and light-independent. When cells were subcultured through serial transfer in the absence of inorganic phosphate, the production of polymetaphosphate granules was almost completely stopped. After the serial transfer, these cells showed little or no endogenous release of inorganic phosphate or release from added polymetaphosphate. Using such polymetaphosphate-free cells, it has been possible to demonstrate photosynthetic phosphorylation by the naturally occurring photosynthetic macro-molecules isolated from this organism. These particles, of about 100 A in diameter with a molecular weight of approximately 1.5 million, are by far the simplest functional naturally occurring photosynthetic electron-transport units thus far described.

Adenosine Diphosphate↗

PubMed2003

Automated extraction and quantification of human cytomegalovirus DNA in whole blood by real-time PCR assay.

The measurement of human cytomegalovirus (HCMV) DNA in blood is becoming the standard method for monitoring HCMV infection in immune-suppressed and unsuppressed patients. As various blood compartments can be used, we have compared the HCMV DNA measured in whole blood (WB), peripheral blood leukocytes (PBL), and plasma by real-time PCR. We tested 286 samples: HCMV DNA was extracted automatically from WB and PBL with the MagNA Pure instrument (Roche Molecular Biochemicals) and manually from plasma samples. The HCMV DNA from WB, PBL, and plasma was measured by real-time Light Cycler PCR. Primers and probe were located in the UL 83 region. HCMV DNA was detected more frequently in WB (88.5%) than in the PBL (65.7%) (P < 0.0001) or the plasma (55.2%) (P < 0.0001). There was a good correlation between the positive results in WB and in PBL (r = 0.68; P < 0.0001), and 3.15 log(10) genome copies in 200000 PBL, equivalent to the threshold value of 50 pp65-positive polymorphonuclear cells per 200000 leukocytes, was equivalent to 3.4 log(10) genome copies in 200 microl of WB. WB was shown to be suitable for automated extraction and the quantitation of HCMV DNA by real-time Light Cycler PCR by analysis of serial samples from representative patients of various populations. This system may be very useful for monitoring of immune-suppressed and unsuppressed patients.

Adult↗

PubMed1972

Acquisition of sequences homologous to host deoxyribonucleic acid by closed circular simian virus 40 deoxyribonucleic acid.

The synthesis of closed circular simian virus 40 (SV40) deoxyribonucleic acid (DNA) containing sequences homologous to host cell DNA depends upon the conditions under which the cells are infected. When BS-C-1 monkey cells were infected with non-plaque-purified virus at low multiplicity of infection [MOI, 0.032 plaque-forming units (PFU)/cell], little, if any, of the SV40 DNA extracted from the infected cells hybridized to host DNA; but when increasingly higher multiplicities were used (in the range 0.16 to 3,000 PFU/cell), an increasingly greater amount of the extracted SV40 DNA hybridized to host DNA. The same effect was observed when the closed circular SV40 DNA was extracted from purified virions (grown at low and high MOI) rather than from the infected cell complex. When the cells were infected at high MOI with plaque-purified virus (11 viral clones were tested), none of the SV40 DNA extracted from the cells hybridized detectably with host cell DNA. However, plaque-purified virus that was serially passaged, undiluted, induced the synthesis of virus DNA which again showed extensive homology to host DNA. It is suggested that, under certain circumstances, recombination occurs between viral and host DNA during lytic infection which results in the incorporation of host DNA sequences into closed circular SV40 DNA.

Animals↗

PubMed1983

Extrahypothalamic brain luteinizing hormone: characterization by radioimmunoassay, chromatography, radioligand assay and bioassay.

We have recently reported that luteinizing hormone (LH) is present in the hypothalamus of rats. It has chromatographic and biologic characteristics similar to pituitary LH. In this report we focus on extrahypothalamic LH that is widely distributed in the rodent central nervous system. This material has a chromatographic profile similar to that of pituitary LH. Serial dilution of this material is parallel with dilutions of rat pituitary LH in the immunoassay. Brain extracts are active in the testis LH radioligand receptor assay and in the rat interstitial cell testosterone secretion bioassay. Prior incubation of extract with LH antibody significantly attenuated both of these activities. Thus, extrahypothalamic LH has immunologic, chromatographic, and biologic characteristics similar to hypothalamic and pituitary LH.

Animals↗

PubMed1985

Application of a unique monoclonal antibody as a marker for nonproliferating subpopulations of cells of some tissue.

A monoclonal antibody (clone S-30), directed to a protein of 57,000 daltons, was developed from the fusion of mouse myeloma cells and the spleen cells of mice injected with cytoskeletal extracts of fibroblasts that have been aged in in vitro culturing conditions according to a schedule of serial passaging (Cristofalo VJ, Charpentier R: J Tissue Culture Meth 6:117, 1981; Wang E: J Cell Biol, submitted). The staining activity of S-30 antibody was observed exclusively in the nuclei of nonproliferating senescent fibroblasts, but not in their young counterparts. Immunolocalization of S-30 antibody in frozen tissues from various sites reveals the positive staining reaction in the nuclear envelope region in those cells that are at the final stage of differentiation and are no longer replicating. These tissue sites include epithelial cells of the suprabasal layer of epidermis, hair sheath, and tongue, a subpopulation of fibroblasts in the dermis, chondrocytes, hepatocytes, and cells of cardiac muscle. The absence of S-30 staining activity was noted in tissues such as simple epithelium located in the gastrointestinal tract and kidney, and keratinocytes in the basal layer. These results suggest that the S-30 antibody can be used as a marker for nonproliferating cells both in cultured fibroblasts and in some tissues. It seems that the mechanism that controls the cessation of cell proliferation is related, in part, to the postmitotic expression of the 57,000 dalton protein.

Aged↗

PubMed1994

Detection of African horsesickness virus by reverse transcriptase polymerase chain reaction (RT-PCR) using primers for segment 5 (NS1 gene).

The reverse transcription followed by the polymerase chain reaction (RT-PCR) technique was applied to the detection of African horsesickness virus (AHSV) using primers specific for attenuated AHSV serotype 4 segment 5 (NS1 gene). Total RNA which contains both messenger RNA and genomic dsRNA was extracted by the acid guanidinium-phenol-chloroform method from the AHSV infected Vero cells and was used as templates to optimize the RT-PCR. A pair of primer (NP2-NP32) amplified the product of the expected size from all serotypes of attenuated AHSV when four pairs of primers were tested. Using this primer pair, no RT-PCR product was detected from the RNA samples extracted from ten other orbiviruses infected cells and their virions. In addition, RT-PCR using a serial dilution of RNA samples suggested that AHSV was efficiently detected from 1 to 2 cells of the cell monolayer infected with 10(6) TCID50 of AHSV. The RT-PCR concerning with total RNAs of AHSV NS1 gene was found to be a specific and sensitive method for the detection of AHSV.

African Horse Sickness↗

PubMed1976

Long-term study of pulp capping in monkeys with three agents.

The pulps of 40 permanent teeth of two monkeys were mechanically exposed and contaminated with adjacent saliva or plaque for 3 to 5 1/2 hours. The pulps were then capped with either Keflin (an antibiotic), Durelon (a polycarboxylate cement), or Dycal (a calcium hydroxide compound). Varnish and then amalgam were inserted. Each monkey received at least one dose of Procion red H-8BS vital dye. The teeth were extracted from one monkey 23 months after capping and from the other monkey 29 months after capping. Serial, decalcified, 7 mum-thick paraffin sections were prepared. Alternate slides were stained with hematoxylin ane eosin. Unstained sections were examined for Procion labeling, and selected slides were stained by the Brown-Brenn method for bacteria. Of 13 teeth capped with Keflin, only 4 responded satisfactorily. All of the Dycal and Durelon-capped pulps were successful at both time intervals. All of the Dycal-capped pulps showed complete bridging and no inflammation or pulp obliteration. Bridging was complete in only three of the Durelon-capped pulps. The findings of this study support the findings in our previous study3 and further substantiate the effectiveness of Dycal as a pulp-capping agent. The often-repeated claim that calcium hydroxide compounds exert a persistent stimulating effect on the pulp resulting in its eventual obliteration was not supported. Durelon is not recommended for pulp capping since the material apparently lacks an antibacterial effect and does not stimulate reparative dentinogenesis at the exposure site. The low rate of satisfactory responses of pulps capped with Keflin, as used, precludes its use of pulp capping.

Acrylates↗

PubMed1980

Niacin requirement for growth of axenic Entamoeba histolytica.

Niacin (nicotinic acid) was found to be essential for the cultivation of axenic Entamoeba histolytica. This vitamin requirement was also satisfied by nicotinamide. Panmede liver digest, a source of vitamins in the axenic medium was replaced with a dialyzed hot water extract of homogenized whole liver, supplemented with various growth factors. Cultured medium made with the liver extract and not supplemented with niacin failed to support continued multiplication of E. histolytica, but did support serial subculture when niacin was added. The concentration of added niacin required to achieve maximum growth was about 1 microgram per ml of medium. This is the first demonstration of a niacin requirement by the organism.

Animals↗

PubMed2002

[Three-dimensional projection display of striatum dopamine function measured by PET].

[(11)C]CFT and[(11)C]raclopride images obtained by positron emission tomography (PET) are used to evaluate pre-synaptic dopamine transporter availability and post-synaptic dopamine D(2) receptor binding, respectively. A combined study with these tracers is useful for the differential diagnosis of Parkinson's disease. We generated three-dimensional (3D) animations of striatum PET images for the diagnosis of Parkinson's disease. Brain images of a normal subject and a typical Parkinson's disease patient with[(11)C]CFT and[(11)C]raclopride were obtained using a PET camera. Three-dimensional animations were generated from serial maximum intensity projection (MIP) images created by gradually changing the projection angle. Furthermore, the striatum images extracted from brain data were superimposed over a brain surface magnetic resonance (MR) image that was created by the volume-rendering method, and 3D animations were similarly generated. The present 3D animations were clinically useful for the differential diagnosis of brain diseases, because we were able to observe distributions of[(11)C]CFT and[(11)C]raclopride from any angle and to grasp at a glance the regional differences of distributions in reference to anatomical landmarks.

Corpus Striatum↗

PubMedDate not supplied

Effect of peroxide-based bleaching agents on enamel ultimate tensile strength.

This study evaluated the effects of peroxide bleaching regimens on the ultimate tensile strength (UTS) of human enamel. A resin composite block was built-up on the bonded occlusal surface of 14 extracted, sound, erupted third molars to enable posterior preparation for the microtensile test. The bonded teeth were serially sectioned in a buccal-lingual direction into approximately 0.7-mm thick slices. Each slice was trimmed with a fine diamond bur to reduce the area of the buccal, internal slope of the cusps to a dumb-bell shape with a cross-sectional area of less than 1 mm2. The samples were randomly divided into seven groups (n = 10): unbleached control group and bleached groups treated with six bleaching regimens. The specimens were tested in tension at 0.5 mm/minute and the data were analyzed by ANOVA and Tukey test. Specimens from the control group presented 51.3 +/- 8.6 MPa, while the UTS of bleached enamel ranged from 22.0 +/- 5.6 to 36.3 +/- 9.1 MPa. All bleaching procedures significantly reduced enamel UTS (p < 0.05). Differences were also observed among treatments. The results suggested that bleaching regimens can significantly reduce enamel UTS.

Analysis of Variance↗

PubMedDate not supplied

[Principles of dental treatment of handicapped children in general anaesthesia].

The long experience and practice in the treatment of handicapped children determined the appropriate general type of anaesthesia as well as the method and order of dental operations under general anaesthesia. A wide variety of dental treatment in such conditions concerns the "serial work per quadrants", strong measures of preventive extension and pulp protection, and selection of indications for extraction of teeth. It is emphasized that during the postoperative period frequent control examinations and intensified preventive measures by parents and dentists are necessary.

Anesthesia, Dental↗

PubMed1977

Radioimmunoassay of vasoactive intestinal polypeptide (VIP) in plasma.

A sensitive and specific radioimmunoassay for vasoactive intestinal polypeptide (VIP) has been developed, which can detect 3.3 pmol x L-1 of the peptide in plasma. Antisera to highly purified porcine VIP coupled to albumin were raised in eight rabbits. The final dilution, the avidity, and the specificity of each antiserum were determined. 125I-VIP served as label, and highly purified porcine VIP was used as standard. Separtation of antibody-bound and free VIP was achieved by plasma-coated charcoal. Nonspecific interference withe assay system was excluded by extraction of plasma samples with ethanol. The reliability of the assay was investigated by recovery experiments, by serial dilution of plasma samples with high concentration of endogenous VIP, and by immunosorption. The within-and between assay reproducibility at a concentration of 18.3 pmol x L-1 was 1.6 and 2.3 pmol x L-1 (1 S.D.), respectively. Median fasting concentration of VIP in plasma from 74 normal subjects was 7.3 pmol x L-1 (range: 0-20.0 pmol x L-1).

Adolescent↗

PubMed1985

Technetium-99m DADS complexes as renal function and imaging agents: II. Biological comparison with iodine-131 hippuran.

To find a 99mTc agent with a high renal extraction efficiency similar to [131]hippuran, 21 analogs of DADS were labeled and evaluated. Preliminary screening by serial gamma camera imaging in rabbits showed that most analogs had a higher liver uptake and/or slower renal clearance, than hippuran. Three agents (P-DADS, AP-DADS, and CAP-DADS), exhibiting a relatively rapid blood clearance and lower liver uptake in the rabbit, were studied in greater detail in comparison with hippuran and CO2-DADS-A, the best analog to date. In the rat, the plasma clearance of the four DADS analogs was slower than that of hippuran. In rats with tubular damage induced by cisplatin, the difference in renal retention at 1 hr compared to controls was much greater with hippuran than with the DADS compounds. In the dog, there was marked hepatic retention of the four DADS compounds. In volunteers, serial posterior images obtained with these [99mTc]DADS complexes showed significant hepatic as well as renal activity. The 1-hr plasma clearance and urinary excretion were much lower than with simultaneously injected hippuran. Although these 99mTc agents are satisfactory for imaging the kidneys, they closely mimic the biodistribution of hippuran only in the rabbit, and not in the rat, dog, or man.

Animals↗

PubMedDate not supplied

Surface antigen on Ehrlich ascites tumor cells.

Ehrlich Ascites Tumor (EAT) may be regarded as an extreme example of a reduced expression of normal histocompatibility antigens which is also reflected in its ability to grow in almost any mouse strain. In this report we discuss some evidence about a specific antigenicity detected on the cell surface of EAT strain maintained by serial transfer in C57BL/6 mice. The antiserum against EAT antigen was prepared by immunizing rabbits with an extract obtained by KCl treatment of EAT cells and then tested for cytotoxicity on EAT cells. The specificity, confirmed by using the indirect immunofluorescence test and the inhibition of cytotoxicity by EAT antigen, was also discussed.

Animals↗

PubMed1978

[Organochlorine insecticide residues in tobacco and tobacco products. 1. Analytical method].

An analysis method for quantitative serial estimation of organochlorine insecticide residues in tobacco is reported. This method is based on an hexane extraction followed by column chromatographic clean-up and preseparation as well as qualitative and quantitative gas chromatographic analysis. The following 16 substances are analyzed: HCB, alpha-HCH, lindane, heptachlor, aldrin, heptachlor epoxide, o,p'-DDE, p,p'-DDE, O,P'-TDE, p,p'-TDE, o,p'-DDT, dieldrin, endosulfan, beta-endosulfan and endosulfan sulfate. In a sample of 5 g of tobacco there can be detected: HCB, alpha-HCH, lindane, heptachlor, aldrin, heptachlor epoxide--o,oo1 ppm; o,p'-DDE, p,p'-DDE, dieldrin--o,oo3 ppm; o,p'-TDE, p,p'-TDE-- o,o1 ppm; o,p'-DDT, alpha-endosulfan, beta-endosulfan--o,o2 ppm; endosulfan sulfate--o,o3 ppm; p,p-DDT--o,o5 ppm.

Chromatography↗

PubMedDate not supplied

Selected uses of enzymes with critical fluids in analytical chemistry.

The use of enzymes coupled with supercritical fluid (SF)-based analytical techniques, such as supercritical fluid extraction (SFE), provides a safer environment platform for the analytical chemist and reduces the use of organic solvents. Incorporation of such techniques not only reduces the use of solvent in analytical laboratories, but it can also lead to overall method simplification and time savings. In this review, some of the fundamental aspects of using enzymes in the presence of SF media are discussed, particularly the influence of extraction (reaction) pressure, temperature, and water content of the extracting fluid and/or the sample matrix. Screening of optimal conditions for conducting reactions in the presence of SF media can be readily accomplished with automated serial or parallel SFE instrumentation, including selection of the proper enzyme. Numerous examples are cited, many based on lipase-initiated conversions of lipid substrates, to form useful analytical derivatives for gas chromatography, liquid chromatography, or SF chromatography analysis. In certain cases, enzymatic-aided processing of samples can permit the coupling of the extraction, sample preparation, and final analysis steps. The derived methods/techniques find application in nutritional food analyses, assays of industrial products, and micro analyses of specific samples.

Animals↗

PubMed2001

A cystatin-based affinity procedure for the isolation and analysis of papain-like cysteine proteinases from tissue extracts.

Cysteine-proteinases (CP) of the papain family can be affinity-adsorbed by egg white cystatin C coupled to Sepharose 4B, thus allowing their selective isolation from either tissue or cultured cell extracts as well as biolological fluids and culture media. CP complexed by immobilized cystatin are further analyzed by means of SDS-PAGE and Western blot followed by serial or parallel immunological detection. The single-step affinity adsorption of papain-like enzymes has the advantage, over immunoprecipitation techniques, of yielding the simultaneous and comprehensive picture of most CP, as both precursor and mature forms, in a given sample. Moreover, cell extraction in the presence of immobilized cystatin ensures a fast complexation of CP, avoiding artifacts, due to conversion, degradation, and, eventually, subtraction of constitutive enzymes from the sample because of their interactions with endogenous inhibitors. This will provide a pattern that might reflect more closely the real CP levels in intact cells. The method may be useful in the field of biochemistry, cell biology, and, possibly, clinical chemistry to perform rapid analyses of papain-like enzymes and to monitor changes in both cellular and extracellular CP profiles along with different physiopathological conditions.

Adsorption↗

PubMed2003

Measurement of inflammatory biomarkers in synovial tissue extracts by enzyme-linked immunosorbent assay.

We developed methods for measuring inflammatory biomarkers (cytokines, chemokines, and metalloproteinases) in synovial biopsy specimens from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Soluble extracts of synovial fragments were prepared with mild detergent and analyzed by enzyme-linked immunosorbent assay (ELISA) for interleukin 1beta (IL-1beta), IL-6, IL-8, tumor necrosis factor alpha (TNF-alpha), and matrix metalloproteinase 3. The optimal detergent was 0.1% Igepal CA-630, which interfered minimally with ELISA detection but extracted 80% of IL-6 from synovial tissue. Upon spiking, 81 to 107% of added biomarkers could be recovered. To determine within-tissue variability, multiple biopsy specimens from each RA synovial extract were analyzed individually. A resulting coefficient of variation of 35 to 62% indicated that six biopsy specimens per synovial extract would result in a sampling error of < or = 25%. Preliminary power analysis suggested that 8 to 15 patients per group would suffice to observe a threefold difference before and after treatment in a serial biopsy clinical study. The previously described significant differences in IL-1beta, IL-6, IL-8, and TNF-alpha levels between RA and OA could be detected, thereby validating the use of synovial extracts for biomarker analysis in arthritis. These methods allow monitoring of biomarker protein levels in synovial tissue and could potentially be applied to early-phase clinical trials to provide a preliminary estimate of drug efficacy.

Arthritis, Rheumatoid↗