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PubMed1989

Partial color constancy of isolated surface colors examined by a color-naming method.

Color samples selected from the OSA Uniform Color Scales set were viewed without any surround. Separate light sources were used to illuminate the samples and to control the state of adaptation of the subject, thereby separating two factors that are normally confounded. A color-naming procedure was used to assess shifts in color appearance produced by altering the spectral distributions of one or both light sources. The results confirm that chromatic adaptation, when it is the only factor operating, can mediate partial color constancy.

Color Perception↗
PubMed1990

Genetic diversity and ecogeographical differentiation among ribosomal DNA alleles in wild and cultivated barley.

DNA from 267 accessions of wild barley from ecologically diverse habitats in Israel and Iran and from 92 accessions of cultivated barley from throughout the world were assayed for the 20 ribosomal DNA (rDNA) spacer-length variants that have been identified in the barley species. These 20 spacer-length variants, which are detectable by Southern blot hybridization, serve as markers of rDNA alleles of two Mendelian loci, Rrn1 and Rrn2. All of the populations of wild barley studied were polymorphic for both loci. In wild barley allele 112 (Rrn1) and allele 107 (Rrn2) behaved as widely adapted wild-type alleles; in our sample of cultivated barley allele 112 also behaved as a wild-type allele but allele 104 was somewhat more frequent than allele 107 in Rrn2. A few other alleles were locally frequent in wild barley. However, most of the 20 alleles were infrequent or rare and such alleles were often associated as "hitchhikers" with one of the wild-type alleles in compound two-component alleles. Allelic and genotypic frequencies differed widely in different habitats in correlation with eight of nine factors of the physical environment. Discrete log-linear multivariate analyses revealed statistically significant associations among alleles of Rrn1 and Rrn2. It was concluded that natural selection acting differentially on various rDNA alleles plays a major role in the development and maintenance of observed patterns of molecular and genetic organization of rDNA variability.

Alleles↗
PubMed1996

An ultrastructural and cytochemical study of the mesonephros of Rana esculenta during activity and hibernation.

The biological cycle of most amphibians undergoes seasonal variations. In this study, we investigated the mesonephros of Rana esculenta during active life and the natural hibernation period. The ultrastructural morphology of the different tracts constituting the nephron was analysed. Moreover, to evaluate the effect of seasonal temperature variations on the mesonephros function, the activity of some enzymes linked to membrane transport and playing regulatory roles in various metabolic pathways was investigated in different tracts of the frog nephron. During hibernation the glomerular filtration barrier appeared thicker than in the active life, lysosomes and paraplasmatic material, mostly glycogen, being accumulated in the proximal and distal tubule cells respectively. Cytoplasmic organelles, i.e., mitochondria, endoplasmic reticulum were observed in segregated areas. At the same time, changes in some enzyme activities were noted. The activity of some membrane-transport enzymes (5' nucleotidase and K+-p-nitrophenyl phosphatase) and of energetic metabolism (succinic dehydrogenase) was reduced. Nevertheless the alkaline phosphatase activity was not changed significantly, and this suggests that some metabolic activities were preserved in the hibernating samples. These results indicate morpho-functional adaptations of the kidney cells that preserve their role in osmoregulation and some metabolic processes, even during unfavourable seasons.

4-Nitrophenylphosphatase↗
PubMed1983

Preparation of peptides containing any desired amino acid: methionyl peptides of bovine rhodopsin.

A general method is described which allows the identification and preparation of peptides containing any amino acid of interest. The method has been applied to isolation of the methionyl peptides from a peptic digest of oxidized bovine rhodopsin. The peptide digestion mixture is first partially separated by ion exchange column chromatography. Location of peptides containing the desired amino acid is performed by amino acid analysis of acid hydrolyzed column fractions by high voltage paper electrophoresis. Peptides are further purified and prepared by peptide mapping, elution, and amino acid analysis using inexpensive high capacity techniques. Peptide sequencing is performed by a manual dansyl-Edman method well adapted for rapidly processing large numbers of samples. The methods are particularly well suited for detection and preparation of peptides containing amino acids for which there is no specific detection method.

Amino Acid Sequence↗
PubMed2001

Culture of bovine bone marrow progenitor cells in vitro.

In vitro methylcellulose cultures of bovine bone marrow progenitor cells were developed. An existing technique described for bovine species was compared to a method for human tissue and further adapted during subsequent experiments. Bovine bone marrow samples were collected at the slaughterhouse, and mononuclear cells were separated by gradient centrifugation (1.077 g/ml specific density and 400 g). The use of 3% bovine leucocyte-conditioned medium, produced by stimulation of blood lymphocytes with 4 microg/ml concanavalin A and harvested on day 4 of culture, gave better results than the use of supernatant of the human bladder carcinoma 5637, which is widely used in human bone marrow cultures. However, bovine leucocyte-conditioned medium was not added to erythroid cultures because inhibitory effects were observed. Erythroid colonies were stimulated with erythropoietin, and hemin was added to enable microscopic identification. Reduced oxygen tension was necessary to induce growth of erythroid colonies. This was not necessary for myeloid cultures. In conclusion, the results of this study show that the growth of myeloid and erythroid colonies in methylcellulose-based medium requires different culture conditions, which are different from the culture conditions for human cells.

Animals↗
PubMed2005

Multi-allergen screening immunoassay for the detection of protein markers of peanut and four tree nuts in chocolate.

A multiresidue enzyme immunoassay was developed to check for the presence of markers of peanut, hazelnut, almond, cashew and Brazil nuts in a single run. The assay was designed under the competitive indirect format and adapted for screening purposes applied to chocolate samples. The limit of detection for this assay was below 1 microg g-1 protein for each allergenic food. In most cases, the high specificity of the antibodies used allowed the identification of each particular allergenic food with no possible confusion. This assay was proven to be useful as part of an analytical procedure involving the identification of the unknown allergenic food among peanut and other tree nuts in recalled samples before the application of a quantitative technique to determine the level of cross-contamination.

Allergens↗
PubMed1987

A solid-phase receptor binding assay for 125I-hCG.

A solid-phase radioligand-receptor assay (RRA) to measure the binding of 125I-labelled human chorionic gonadotropin (125I-hCG) to target cell membranes has been developed. The binding of 125I-hCG to membranes immobilized on the wells of microtitration plates reached a maximum at about 3 hours at 37 degrees C, was saturable, displayed a high affinity (Ka = 2.4 X 10(9) M-1) and was specifically inhibited by unlabelled hCG. In comparison with RRAs carried out with membranes in suspension, the solid-phase RRA is significantly simpler and much faster to perform as it avoids centrifugation or filtration procedures. The solid-phase RRA was adapted profitably to process large numbers of samples at the same time. It proved particularly useful as a screening assay to detect anti-hCG monoclonal antibodies with high inhibitory activity for binding of 125I-hCG to its receptors.

Animals↗
PubMed1985

A rapid method for comparing monoclonal antibodies by limited proteolysis and electrophoresis.

A rapid and sensitive method for comparing the primary structure of proteins has been adapted to the study of monoclonal antibodies. Samples were digested with alpha-chymotrypsin in the presence of sodium dodecyl sulfate after which peptide fragments were separated into distinctive banding patterns by polyacrylamide gel electrophoresis. This method could easily detect differences in the primary structure of antibodies with related as well as unrelated binding specificities. In addition, antibody molecules derived by somatic diversification from the same germ line gene segments could be distinguished from one another.

Amino Acid Sequence↗
PubMed1994

Organ-specific modulation of steady-state mdr gene expression and drug resistance in murine colon cancer cells.

BACKGROUND: The major cause of death from cancer is metastases that are resistant to conventional therapies. The resistance of metastatic tumor cells to chemotherapy can be caused by their intrinsic properties, such as increased expression of the mdr genes. PURPOSE: The purpose of our present study was to determine some of the mechanisms by which the organ microenvironment influences the response of tumor cells to chemotherapy. METHODS: Murine CT-26 colon cancer cells growing in continuous culture (parental cells) were harvested and injected subcutaneously into the lateral flank (to produce subcutaneous tumors) or the lateral tail vein (to produce experimental lung metastases) of 10 8-week-old syngeneic male BALB/c mice. Seven days after tumor-cell injection, the mice were given intravenous injections of either doxorubicin (10 mg/kg) or 0.9% NaCl (controls). This in vivo injection was repeated 7 days later. Mice with subcutaneous tumors and lung metastases were killed by cervical dislocation on day 21, and tumor samples from control mice were harvested and adapted to culture. The sensitivity of the cultured cells to doxorubicin and fluorouracil (5-FU) was determined at multiple time points. Levels of mdr-1 DNA were measured by slot-blot and Southern-blot analyses. mdr mRNA expression levels were measured by Northern-blot analysis using mdr-1- and mdr-3-specific hybridization probes, and P-glycoprotein level was determined by fluorescence-activated cell sorting using different monoclonal antibodies. RESULTS: Treatment with doxorubicin produced 80% growth inhibition of CT-26 subcutaneous tumors but had little effect on the number (and size) of experimental lung metastases. Collectively, the results suggest that the multidrug-resistant phenotype developed in CT-26 cells growing in the lung environment. Cultures established from lung metastases were initially resistant to doxorubicin (but not to 5-FU) and showed elevated expression of mdr-1 mRNA transcripts and P-glycoprotein. This resistance could be overcome by verapamil and disappeared after 21 days in culture. No mdr gene amplification was detected. The expression level of mdr-specific mRNA (predominance of mdr-1) and P-glycoprotein was directly associated with resistance to doxorubicin. CONCLUSIONS: Results of this study have demonstrated that the in vivo sensitivity of murine CT-26 colon carcinoma cells to doxorubicin depends on the organ environment. The organ environment can influence the P-glycoprotein-mediated multidrug-resistant phenotype in tumor cells, and the increased expression of P-glycoprotein is transient; once removed from the environment (lung), the cell's resistance reverts to that of the sensitive parent cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Mutagenicity of the potent rat hepatocarcinogen 6BT to the liver of transgenic (lacI) rats: consideration of a reduced mutation assay protocol.

6-(p-dimethylaminophenylazo)benzothiazole (6BT) is an unusually potent rat hepatocarcinogen, producing large malignant liver tumours after only 2-3 months of dietary administration in a riboflavin-deficient diet. This azocarcinogen has been evaluated in a Big Blue F344 transgenic rat (lacI) gene mutation assay. In a reproduction of the early stages of the carcinogenesis bioassay of this agent, rats were maintained on a riboflavin-deficient diet and were given 10 consecutive daily doses of 6BT (10 mg/kg) by oral gavage. The animals were killed and the livers examined 11 days after the final dose. The livers of 6BT-treated rats showed evidence of hepatocellular hypertrophy in centrolobular areas, with some indication of an increased incidence of mitotic figures. An approximately 10-fold increase in the mutation frequency of DNA isolated from an aliquot of the combined liver homogenates of 6BT-treated rats was observed over that obtained from an equivalent aliquot from control animals. Examination of DNA samples isolated from the livers of individual animals confirmed that 6BT was mutagenic in Big Blue rat livers. These data extend the sensitivity of this transgenic assay to include azo hepatocarcinogens. The determination of mutation frequencies using pooled tissue samples represented a major resource-saving adaptation of the assay protocol in the present study; the general advantages and disadvantages of this practice are discussed.

Animals↗
PubMed1996

Preference for guanosine at first codon position in highly expressed Escherichia coli genes. A relationship with translational efficiency.

The variation in base composition at the three codon sites in relation to gene expressivity, the latter estimated by the Codon Adaptation Index, has been studied in a sample of 1371 Escherichia coli genes. Correlation and regression analyses show that increasing expression levels are accompanied by higher frequencies of base G at first, of base A at second and of base C at third codon positions. However, correlation between expressivity and base compositional biases at each codon site was only significant and positive at first codon position. The preference for G-starting codons as gene expression level increases is discussed in terms of translational optimization.

Amino Acids↗
PubMed1984

Correlation between skin glycosylation and glycaemic control in human diabetes.

Glycosylation of human skin has been measured by an adaptation of the thiobarbituric acid reaction on samples obtained mainly at autopsy from normal and diabetic subjects. The mean (+/- 1 SD) glycosylation of skin from 33 normal subjects was 0.072 +/- 0.012 mumol fructosamine/100 mg wet weight tissue compared with 0.127 +/- 0.034 mumol (P less than 0.01) in the 10 diabetic subjects. There was a correlation between mean blood glucose levels in the 6 months before death and skin glycosylation in the diabetics, (r = 0.71, P = 0.025), but no correlation between age, sex, or the presence or absence of diabetic complications and skin glycosylation. Skin glycosylation in samples from four non diabetics was increased from 0.075 +/- 0.015 to 0.126 +/- 0.01 mumol fructosamine/100 mg wet weight tissue (P less than 0.01) after incubation with 50 mmol/l glucose solution for 7 d at 37 degrees C.

Adolescent↗
PubMed1993

Comparison of two anesthesia techniques on perioperative insulin response to i.v. glucose infusion in children.

Perioperative blood glucose and insulin levels were measured in children (1-9 years of age) randomly assigned to two groups according to anesthesia technique, general anesthesia (group GA) or general anesthesia combined with regional anesthesia (group RA). Children in the GA group (n = 10) received halothane and opioids, while children of the RA group received epidural anesthesia with bupivacaine (0.25%) and adrenaline combined with halothane anesthesia (n = 10). Children in both groups received 2.5% dextrose in 0.4 N saline administered by volumetric infusion pumps throughout the study period, the infusion rate being adapted to the child's age. Blood samples for glucose and insulin determinations were obtained: at induction, at the end of surgery, and 30, 60 and 120 min after surgery. In response to an identical glucose load, blood glucose levels increased significantly in both groups (P < 0.001), while no differences between groups were observed. Insulin levels did not change significantly postoperatively in the GA group (P = 0.058), while a significant increase was observed in the RA group (P < 0.001). Insulin/blood glucose ratio increased significantly only in the RA group (P < 0.05). The higher insulin secretion in response to glucose infusion in the RA group compared to the GA group may indicate an increased peripheral insulin resistance after regional anesthesia or, more likely, this secretion may be beneficial in contributing to improve postoperative nitrogen balance.

Anesthesia, Conduction↗
PubMed2005

Molecular characterization of cyanobacterial diversity in a shallow eutrophic lake.

We have studied the diversity of pelagic cyanobacteria in Lake Loosdrecht, The Netherlands, through recovery and analysis of small subunit ribosomal RNA gene sequences from lake samples and cyanobacterial isolates. We used an adapted protocol for specific amplification of cyanobacterial rDNA for denaturing gradient gel electrophoresis (DGGE) analysis. This protocol enabled direct comparison of cyanobacterial community profiles with overall bacterial profiles. The theoretical amplification specificity of the primers was supported by sequence analysis of DNA from excised DGGE bands. Sequences recovered from these bands, in addition to sequences obtained by polymerase chain reaction (PCR) and cloning from lake DNA as well as from cyanobacterial isolates from the lake, revealed a diverse consortium of cyanobacteria, among which are representatives of the genera Aphanizomenon, Planktothrix, Microcystis and Synechococcus. One numerically important and persistent cyanobacterium in the lake, Prochlorothrix hollandica, appeared to co-occur with an unknown but related species. However, the lake is dominated by filamentous species that originally have been termed 'Oscillatoria limnetica-like'. We show that this is a group of several related cyanobacteria, co-occurring in the lake, which belong to the Limnothrix/Pseudanabaena group. The available variation among the coexisting strains of this group can explain the persistent dominance of the group under severe viral pressure.

Bacterial Typing Techniques↗
PubMed1997

New growth hormone assays: potential benefits.

Three recently published new assays are described for the measurement of growth hormone (GH). Two of these--the eluted stain assay (ESTA) and the immunofunctional assay (IFA)--have been developed to measure the bioactivity of GH, rather than the immunoactivity as measured by conventional radioimmunoassays (RIAs). The third assay--the 22 kDa exclusion assay (22 k GHEA)--is designed to measure the concentrations of the different isoforms of GH present in the circulation. The ESTA is a variant of the Nb2 bioassay for lactogenic hormones, but has been adapted for specific GH measurement in serum samples. It has a lower detection limit than previous bioassays and permits the quantification of GH in large series of samples. The IFA uses a binding-site-specific antibody in combination with GH-binding protein (GHBP) in order to quantify only those GH molecules that are able to dimerize the extracellular domain of the GH receptor (GHBP), which is a prerequisite for GH signal transduction in target cells. The IFA is as convenient to use as immunoassays and can be employed routinely for GH determinations. The clinical usefulness of the 22 k GHEA has not been established, but it should provide a means of augmenting our understanding of the regulation of GH and its various isoforms. Once the ESTA bioassay or the IFA become commercially and widely available, either could replace the RIA as the standard reference method for measuring GH, as both more closely reflect the biologically active proportion of GH in serum samples than that measured by RIA.

Animals↗
PubMed2001

Combination of competitive quantitative PCR and constant-denaturant capillary electrophoresis for high-resolution detection and enumeration of microbial cells.

A novel quantitative PCR (QPCR) approach, which combines competitive PCR with constant-denaturant capillary electrophoresis (CDCE), was adapted for enumerating microbial cells in environmental samples using the marine nanoflagellate Cafeteria roenbergensis as a model organism. Competitive PCR has been used successfully for quantification of DNA in environmental samples. However, this technique is labor intensive, and its accuracy is dependent on an internal competitor, which must possess the same amplification efficiency as the target yet can be easily discriminated from the target DNA. The use of CDCE circumvented these problems, as its high resolution permitted the use of an internal competitor which differed from the target DNA fragment by a single base and thus ensured that both sequences could be amplified with equal efficiency. The sensitivity of CDCE also enabled specific and precise detection of sequences over a broad range of concentrations. The combined competitive QPCR and CDCE approach accurately enumerated C. roenbergensis cells in eutrophic, coastal seawater at abundances ranging from approximately 10 to 10(4) cells x ml(-1). The QPCR cell estimates were confirmed by fluorescent in situ hybridization counts, but estimates of samples with <50 cells x ml(-1) by QPCR were less variable. This novel approach extends the usefulness of competitive QPCR by demonstrating its ability to reliably enumerate microorganisms at a range of environmentally relevant cell concentrations in complex aquatic samples.

Animals↗
PubMed1967

Mode of filamentous growth of Leucothrix mucor in pure culture and in nature, as studied by tritiated thymidine autoradiography.

Mode of growth of Leucothrix mucor filaments was measured by autoradiography with tritiated thymidine. Studies were performed on L. mucor in pure cultures in free suspension, as an epiphyte of pure cultures of the red alga Antithamnion sarniense, and as an epiphyte of red algae in the sea. Statistical analyses of the distribution of growing cells was done by use of the nonparametric One-Sample Runs Test and a Cluster analysis adapted from quadrat analyses of plant ecologists. No evidence of preferential growth at base or tip of L. mucor filaments was obtained in any of these studies. However, in nature, but not in the laboratory, there were regions of L. mucor filaments which were nongrowing or dormant. Such nongrowing regions could incorporate tritiated glucose.

Autoradiography↗
PubMed1978

Acetylation of peptidyl-tRNA on rat liver polyribosomes.

Some properties of the known NH2-terminal acetylation of peptidyl-tRNA on rat liver polyribosomes were studied. Polyribosomes were incubated with [3H]acetyl-CoA and the products were separated into protein and peptidyl-tRNA fractions by an Ecteola-cellulose column procedure adapted for rapid, routine analysis of many samples, so that the characteristics of acetylation of the two fractions could be determined. Extraction of the polyribosomes with 0.5 MKC1--5mM Mg(OAc)2 solubilized acetyltransferase activity which could catalyze histone acetylation, and the extract when added back to the extracted could catalyze only a slight acetylation of peptidyl-tRNA free in solution isolated from polyribosomes by extraction with EDTA. These observations form the basis of any assay specific for the acetyl-CoA:peptidyl-tRNA N-acetyltransferase which is associated with rat liver polyribosomes.

Acetyl Coenzyme A↗