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PubMed1986

Human primary colon carcinomas xenografted into nude mice. I. Characterization of plasminogen activators expressed by primary tumors and their xenografts.

Analysis was made of plasminogen activator (PA) activities present in 0.125% Triton X-100 extracts of human primary colon carcinomas and of their respective serial subcutaneous xenografts in nude mice. A correlation between tumor invasiveness and PA expression was observed in that primary tumors exhibiting clearly invasive growth patterns demonstrated high concentrations of PAs while subcutaneous xenografts, exhibiting noninvasive pseudobenign growth, contained very low levels of PA activity. The decrease in fibrinolytic activity observed in subcutaneous xenografts was not due to an increase in inhibitors of fibrinolytic activity. Immunologic characterization of PAs in tumor extracts showed that over 90% of human PA activity was of the urokinase type. Furthermore, tumor-derived urokinase was shown to be present in a proenzyme form. It was resistant to diisopropyl fluorophosphate (DFP) and was not inhibited by purified PA inhibitor. However, after its activation into urokinase by plasmin, it was completely inhibited by DFP and PA inhibitor.

Adenocarcinoma↗

PubMed1985

Uterine and whole body oxygen extractions in the pregnant rabbit under chronic steady-state conditions.

Seventeen pregnant rabbits were studied under conscious, unstressed conditions after catheterization of the right ventricle (RV), a femoral artery (A), and a uterine vein (UV). Respiratory gas tensions, pH, and oxygen saturations and contents were determined serially throughout the latter half of gestation. The uterine coefficient of oxygen extraction increased with gestational age (R = 0.86) and became 60.6% +/- 0.3% during the last 4 days of pregnancy. To compare uterine perfusion with whole body perfusion in relationship to oxygen demands, the (Ao2 - UVo2/Ao2 - RVo2) ratio was computed. The ratio was greater than or equal to 1 after 20 days of gestation, which demonstrated that in regard to oxygen demands the rabbit uterus is relatively underperfused compared to the rest of the maternal organism in the last part of pregnancy. A comparison with analogous data in other species demonstrates that the pregnant rabbit, like the guinea pig, has a much lower rate of uterine blood flow than does the pregnant sheep. These interspecies differences in the perfusion rate and oxygen extraction of the pregnant uterus are related to differences in placental structure.

Animals↗

PubMed2006

Protein extraction from formalin-fixed, paraffin-embedded tissue sections: quality evaluation by mass spectrometry.

A satisfactory protocol of protein extraction has been established based on the heat-induced antigen retrieval (AR) technique widely applied in immunohistochemistry for archival formalin-fixed, paraffin-embedded (FFPE) tissue sections. Based on AR, an initial serial experiment to identify an optimal protocol of heat-induced protein extraction was carried out using FFPE mouse tissues. The optimal protocol for extraction of proteins was then performed on an archival FFPE tissue of human renal carcinoma. FFPE sections were boiled in a retrieval solution of Tris-HCl containing 2% SDS, followed by incubation. Fresh tissue taken from the same case of renal carcinoma was processed for extraction of proteins by a conventional method using radioimmunoprecipitation assay solution, to compare the efficiency of protein extraction from FFPE tissue sections with extraction from fresh tissue. As a control, further sections of the same FFPE sample were processed by the same procedure without heating treatment. Evaluation of the quality of protein extracted from FFPE tissue was done using gel electrophoresis and mass spectrometry, showing most identified proteins extracted from FFPE tissue sections were overlapped with those extracted from fresh tissue.

Animals↗

PubMed1986

Ectopic G-29 and G-37 glucagon secretion by hypercalcemic infantile renal tumors.

Four hypercalcemic infantile renal tumors were shown to secrete glucagon-like peptides. These unusual tumors were histologically classified as rhabdoid tumors of the kidney (3 cases) and a cellular mesoblastic nephroma (1 case). Elevated G-29 and G-37 glucagon levels were detected in the plasma and tumor extracts as well as in the supernatants of cultured tumor explants. Three of these tumors were heterotransplanted into the nude mice and serially passaged from a mouse to another. The glucagon level decreased in the transplanted tumor extracts with the number of passage.

Animals↗

PubMed1980

Aetiology of jaagsiekte: transmission by means of subcellular fractions and evidence for the involvement of a retrovirus.

Jaagsiekte (ovine pulmonary adenomatosis) was transmitted to new-born lambs by inoculation of the microsomal fraction of a cytoplasmic extract of cultured tumour cells or tumour tissue. Various treatments of the biologically active fraction were carried out to differentiate between various classes of possible aetiological agents. The results obtained suggested the involvement of a membrane-associated RNA containing virus. Reverse transcriptase activity dependent on Mg++ was subsequently demonstrated in these extracts and in lung exudate, and was shown to be associated with particles banding at a density 1,175 in sucrose gradients. These characteristics, as well as the appearance of the particles in the electron microscope, are similar to those reported for Type B and Type D retroviruses. Serial transmissions of jaagsiekte over a number of years, using cytoplasmic extracts and purified virus, strongly suggest that this virus is the aetiologic agent of jaagsiekte.

Animals↗

PubMed1986

Response of the corneal endothelium to cataract surgery.

Regional changes in endothelial cell density, morphology, and corneal thickness were serially examined with a specular microscope after intracapsular cataract extraction (four eyes), extracapsular cataract extraction (five eyes), and extracapsular cataract extraction with posterior chamber lens implantation (13 eyes). Regardless of the type of cataract surgery, cell loss and morphologic changes (decreased percentage of hexagonal cells, increased cellular elongation, and increased coefficient of variation) were greatest and occurred within one week in the superior cornea. Similar changes occurred after one month centrally but were minimal inferiorly. Endothelial cell density stabilized, and the morphologic changes resolved within three months in all regions of the cornea. Corneal swelling resolved within one month. Differences between the procedures were noted only in the superior portion of endothelium, where cell loss and morphologic changes were greatest following intracapsular cataract extraction. Also, endothelial wound healing was complete and stable three months after cataract surgery, with or without posterior chamber lens implantation.

Adult↗

PubMed1990

A baboon model for pregnancy-associated antigens (PAPP-A, PP5, PP14).

By radioimmunoassays established on human derived antigens, PAPP-A, PP5 and PP14 immunoreactivity was detected in placental extracts and blood of pregnant baboons. None of the serial dilution curves suggested parallelism between respective human and baboon samples. Based on slopes of regressed logit-log transformed binding data, PAPP-A demonstrated the greatest degree of interspecies immunological crossreactivity. PP14 showed the least conservation of antigenic determinants. Physicochemical characterization on heparin, zinc chelate and bovine thrombin affinity matrices could not distinguish human from baboon-derived antigens. As in the human, baboon PAPP-A and PP5 were not detected in blood of male or non-pregnant animals. PP14 was detected in baboon follicular fluid, and only PP5 immunoreactivity was measured in culture media of baboon embryos. Of the three antigens, PAPP-A was detected in pregnant baboons at about 61 days gestation, that is, 4 weeks before PP5 and PP14. With the exception of PP14 which attained peak concentration at 118 days of pregnancy, PAPP-A and PP5 concentrations were greatest at term. In conjunction with physicochemical and immunological criteria, these physiological kinetics clearly support a role for developing a baboon model to serve for further studies into feto-maternal signals, particularly antigens such as PAPP-A and PP5.

Animals↗

PubMed1998

Three-dimensional reconstruction of approximal subsurface caries lesions in deciduous molars.

The present study examines the three-dimensional (3D) morphology of early approximal subsurface enamel caries lesions and subjacent dentin reactions in deciduous molars. Twenty-three extracted primary molars were embedded in Technovit 9100 and serial sections were cut using a saw microtome. Forty approximal lesions were found and investigated using polarized light microscopy for the identification of the different zones of the caries lesion. These zones were then reconstructed three-dimensionally using computer-aided 3D reconstruction methods and the dimensions and volumes of the translucent zone, the body of the lesion, and the dentin lesion were calculated. The enamel demineralization index was defined as the ratio between the translucent zone and the body of the lesion, whereas the enamel-dentin demineralization index was defined as the volumetric ratio of the early dentin lesion to the body of the enamel lesion. The 3D reconstruction of the lesions showed extremely heterogeneous micromorphological features of zone profiles. In enamel lesions, the results demonstrated a decreasing enamel demineralization index with increasing size of the lesion, which indicates a high risk of further caries progression. The enamel-dentin demineralization index indicated, in 5 out of 17 dentin lesions, a high risk of further caries progression. Computer-assisted 3D reconstruction and volumetric assessment of initial caries lesions in deciduous molars represents a valuable methodology in pathogenesis studies, which may lead to a better clinical understanding of caries progression.

Dental Caries↗

PubMed2006

Inhibitory effects of Akacid (plus) on growth and aflatoxin production by Aspergillus parasiticus.

The effects of Akacid plus, a novel guanidine-based polymer first introduced as a biocidal and disinfectant agent were studied on Aspergillus parasiticus growth and its aflatoxin (AF) productivity. The fungus was cultured on yeast extract-sucrose (YES) broth in presence of various twofold serial dilutions of 25% Akacid plus (1.5-96 microL/50 mL medium) and then incubated in shaking condition with 150 rev./min at 28 degrees C for 96 h. Based on obtained results, Akacid plus was found to significantly inhibit both growth and aflatoxin B1 (AFB1) synthesis in very low concentrations in a dose-dependent manner. Fungal growth inhibition was determined in the range of 9.6-99.6% in mycelia exposed to the total concentration range of 1.5-48 microL. A final concentration of 96 microL was necessary to completely inhibit the growth of fungus. Under similar conditions, AFB1 synthesis was found to be strongly inhibited by 8.1-98.0% in presence of 1.5-24 microL Akacid plus with a maximum of 100% by 48 microL concentration. With respect to the unique physico-chemical properties of Akacid plus, its marked inhibitory effects on A. parasiticus growth and its AFB1 synthesis shown for the first time in this study make it a promising candidate for application in prevention programmes of AF contamination of susceptible crops.

Aflatoxin B1↗

PubMed1979

The brain of the horseshoe crab (Limulus polyphemus). III. Cellular and synaptic organization of the corpora pedunculata.

The large, hemispherical mass of the Limulus corpora pedunculata consists of two highly branched lobes, each connected to the protocerebrum by a narrow stalk. About 10(4) afferent fibers enter through the stalks and make diverse, profuse, and often reciprocal contacts with several million Kenyon (intrinsic) cells and one another. The Kenyon cell axonal arborizations converge on a few hundred efferent dendrites. The afferent fiber types can be classified into five types. Type A forms the club-shaped core of glomeruli and circumglomerular annuli, and contains small flat vesicles, suggesting an inhibitory function. Type B terminates with bushy endings in glomeruli and is presynaptic to both Kenyon cells and to Type A terminals. It has clear round vesicles and is the presumptive excitatory input. Type C terminates on other afferents, in glomeruli, and rarely on Kenyon cell bodies, contains angular (neurosecretory) granules and is postulated to impart circadian rhythm. Type D terminates on Kenyon cell somata and the initial neurite segment (but not in glomeruli), and contains dense-cored vesicles. Type E terminates in peduncles on other afferents and Kenyon cell telodendria. It contains dense vesicles. The C, D, and E afferents have reciprocal synaptic connections with Kenyon cell axon terminals. Glomeruli thus receive three different inputs of presumptive inhibitory (A), excitatory (B), and neuromodulatory nature (C). Kenyon cells, increasing in number up to about 1 x 10(8) in the adult, show minor variations in their dendritic pattern and have only one rare variant cell type. Interactions between them occur primarily at their axonal boutons as they crowd around efferent fibers. The latter have large receptive fields, some of their large somata are located within the confines of the corpora pedunculata, and they receive input almost only from Kenyon cells. Numerical and directional details of the circuitry in the corpora pedunculata have been extracted by a combination of light and electron microscopy, serial sectioning, silver staining, and stereology. The corpora pedunculata appear to process primarily the voluminous chemosensory input from the appendages, an assumption that is supported by the major connections of the organ.

Animals↗

PubMed1994

Identification of immunoreactive atrial natriuretic peptide in the gallbladder and bile juice of rabbit, pig and human.

The presence of immunoreactive atrial natriuretic peptide (irANP) in rabbit, pig and human gallbladders was investigated using radioimmunoassay and immunohistochemistry. Serial dilution curves of gallbladder tissue and bile juice extracts were paralleled to the standard curve of atriopeptin III. Gel filtration profiles of gallbladder tissue extracts showed a major peak corresponding to rat pro-ANP. The amounts of irANP in rabbit, pig and human gallbladders were 30.0 +/- 12.3 pg/mg (n = 7), 7.0 +/- 2.0 fg/mg (n = 7) and 17.7 +/- 2.0 fg/mg wet tissue (n = 8), respectively. Bile juice was also shown to contain irANP but with small molecular mass. The amounts of irANP in the rabbit, pig and human bile juice were 25.0 +/- 2.0 (n = 7) and 0.50 +/- 0.02 (n = 7), and 1.3 +/- 0.1 pg/ml (n = 8), respectively. The immunohistochemical staining of the rabbit gallbladder tissue revealed the presence of irANP in the luminal epithelium and smooth muscle layer. The amount of irANP was higher in the luminal epithelium than in the rest of the gallbladder tissue from rabbits (0.30 +/- 0.06 vs. 0.01 +/- 0.01 pg/microgram protein, P < 0.01). These findings suggest that ANP may be synthesized and stored in the gallbladder, and may have a role in the regulation of fluid balance and cystic motility.

Animals↗

PubMed1995

Hypovolemic shock and mortality after ingestion of Tripterygium wilfordii hook F.: a case report.

Tripterygium wilfordii Hook F (TWHF) is a kind of Chinese herbal medicine used for 2000 years. It was applied externally for treatment of arthritis and inflammatory tissue swelling in early years. Recently, this drug has been found to have immunosuppressive effects which could successfully induce remission of some autoimmune disorders without obvious adverse effects. Although there are side effects of gastrointestinal upset, infertility and suppression of lymphocyte proliferation, little information about lethal toxicities has been reported. A case is presented here of a previously healthy young man who developed profuse vomiting and diarrhea, leukopenia, renal failure, profound hypotension and shock after ingestion of an extract of TWHF. In addition to his hypovolemic shock, serial electrocardiograms (ECG), cardiac enzyme studies, and echocardiography also showed some evidence of coexisting cardiac damage. He died of intractable shock 3 days after the abuse of TWHF. Further studies of the pathogenesis of peripheral collapse and possible cardiac toxicity, and determination of the therapeutic range of this drug are necessary before it is used extensively.

Adult↗

PubMed1989

Purification and characterization of cytotoxin from the crude extract of Pseudomonas aeruginosa.

Pseudomonas aeruginosa cytotoxin is a protein toxin exhibiting cytotoxic effects on various eukaryotic cells. The toxin was purified from a crude extract of Pseudomonas aeruginosa 158 by trypsin treatment and serial chromatography. The purified cytotoxin was electrophoresed as a single protein band on polyacrylamide gel electrophoresis (PAGE) in the presence or absence of sodium dodecyl sulfate (SDS). The molecular weight of the purified toxin was determined to be 29,000 by SDS-PAGE and gel filtration chromatography in the presence of 6 M guanidine hydrochloride; the isoelectoric point was pH 6.0. The N-terminal amino acid sequence of the purified toxin was determined. The purified toxin showed the strongest cytotoxic effect on rabbit polymorphonuclear leukocytes and diverse degrees of cytotoxic effects on various eukaryotic cells including red blood cells and cultured cells.

Amino Acid Sequence↗

PubMed1994

Differential accumulations of 4,977 bp deletion in mitochondrial DNA of various tissues in human ageing.

Several types of deletions in mitochondrial DNA (mtDNA) have been recently identified in various tissues of old humans. In order to determine whether there are differences in the incidence and proportion of deleted mtDNAs in different tissues during human ageing, we examined the 4,977 bp deletion in mtDNA of various tissues from subjects of different ages. Total DNA was extracted from each of the biopsied tissues and was serially diluted by two-fold with distilled water. A 533 bp DNA fragment was amplified by PCR from total mtDNA using a pair of primers L3304-3323 and H3817-3836, and another 524 bp PCR product was amplified from 4,977 bp deleted mtDNA by identical conditions using another pair of primers L8150-8166 and H13631-13650. The maximum dilution fold of each sample that still allowed the ethidium bromide-stained PCR product (533 bp or 524 bp) in the agarose gel to be visible under UV light illumination was taken as the relative abundance of the mtDNA (wild-type or mutant) in the original sample. By this method, we were able to determine the proportion of deleted mtDNA in human tissues. We found that the 4,977 bp deletion started to appear in the second and third decades of life in human muscle and liver tissues. But the deletion was not detectable in the testis until the age of 60 years. Moreover, the proportion of deleted mtDNA varied greatly in different tissues. Among the tissues examined, muscle was found to harbor higher proportion of deleted mtDNA than the other tissues. The average proportion of the 4,977 bp deleted mtDNA of the muscle from subjects over 70 years old was approximately 0.06%, and that of the liver and the testis was 0.0076% and 0.05%, respectively. These findings suggest that the frequency and proportion of the deleted mtDNA in human tissues increase with age and that the mtDNA deletions occur more frequently and abundantly in high energy-demanding tissues during the ageing process of the human.

Adolescent↗

PubMed2004

Use of serial analysis of gene expression (SAGE) technology reveals new granulocytic markers.

Because of its short life span in blood, its low RNA content and its highly condensed nucleus, the granulocyte was initially considered as a terminally differentiated cell unable to express novel genes. However, mature granulocytes still contain a variety of mRNAs and may respond to external stimuli by rapid and complex changes in gene expression. The present work was undertaken to provide a wider view of the gene expression profile in unstimulated circulating PMNs. We used serial analysis of gene expression (SAGE) adapted to downsized extracts (SADE) to cope with their small mRNA content. As cell samples of the highest degree of purity were crucial for this project, we adapted a magnetic cell sorting method to reach the required high levels of purity (99.55 +/- 0.19%) together with low activation rates (1.37 +/- 0.28). We analyzed 20,787 tags identifying 8547 different transcripts, of which 47.8% were unknown, 34.6% were transcripts of known genes, and 13.8% matched with expressed sequence tags (EST). Highly expressed genes were those involved in cell mobility, diapedesis, cell signaling and destruction of micro-organisms. In addition, this method led to the identification of genes which had not previously been reported in granulocytes. These results could provide new molecular markers and a reliable reference for the investigation of pathologies involving alteration of the granulocyte gene expression profile.

Biomarkers↗

PubMed2006

Non-radioisotope detection of pol sequences of HTLV-1 proviral DNA: Standardisation and sensitivity analysis.

Proviral DNA amplification methods may be used for identification of HTLV-1 infection or in basic virology research. Published standardised methods in this regard usually depend on hybridisation of PCR products with radioisotope-labelled probes. However, this procedure has limited use in routine testing, due to environmental and health risks. The aim was to assess the feasibility of routine use and the accuracy of an alternative detection system that employs an HTLV-1-specific enzyme-labelled probe. For this purpose DNA was extracted from MT-2 cells, quantified and submitted to serial dilution (1:10), starting from 1.2 microg of genomic DNA. Primary and nested PCR amplifications of pol sequences of the HTLV-1 genome were carried out with standardised primers (SK110/111 and POL1.1/3.1). After Southern blotting, two different detection systems were compared, consisting of hybridisation with either 32P- or alkaline phosphatase-labelled SK112 probes. Both detection systems yielded similar results, detecting PCR products generated from 120 pg of DNA (genomic DNA amount equivalent to 20 diploid human cells) after primary and nested PCR. The alkaline phosphatase-labelled detection technique was feasible for the diagnosis of HTLV-1 with the advantage of precluding the handling of radioisotopes.

Alkaline Phosphatase↗

PubMed1998

RT-PCR quantitation of cytokine responses in vivo from specimens containing small numbers of cells during bioimmunotherapy.

A Standard Template method has been developed to carry out semiquantitative RT-PCR analysis on mRNA extracted from small specimens that contain low yields of RNA. Using easily prepared templates (made from previously tested reference specimens), standard calibration curves were generated for each of two cytokine products of interest, specifically IL-10 and IFN-gamma. Also, internal standardization was accomplished by quantitating a well-characterized housekeeping gene (GAPDH). Simple densitometry of the RT-PCR product did not demonstrate sufficient reliability for quantitation since a logarithmic relationship was shown between product and template input. Peritoneal exudate cell specimens that were obtained from ovarian cancer patients during intraperitoneal immunotherapy were utilized for the demonstration of IL-10 and IFN-gamma transcript in vivo. Briefly, this method consists of: (1) template preparation: a PCR product for the cytokine of interest is generated from a previously identified positive specimen, purified and stored at -20 degrees C. (2) RT-PCR: cDNAs are produced from RNA extracted from patient specimens. Replicates of each cDNA and serial dilutions of the corresponding template are amplified with primers specific for each cytokine of interest. (3) Quantitation: photographs are made of the PCR products displayed on an agarose gel and are analyzed by densitometry. Determinations of fold-differences in cytokine transcript expression are normalized to the mRNA content of each specimen (based on the expression of GAPDH).

Ascitic Fluid↗

PubMed1998

Plasma marinobufagenin-like and ouabain-like immunoreactivity in adrenocorticotropin-treated rats.

Recently, an endogenous digitalis-like factor (EDLF) was shown to be stimulated in corticotropin (ACTH) hypertension in the rat. We have shown that mammalian plasma contains a vasoconstrictor Na,K-ATPase inhibitor, which cross-reacts with an antibody to amphibian EDLF, marinobufagenin. In the present experiment, the effect of 8 days of intramuscular ACTH treatment (0.5 mg/kg/day) of male Fisher 344 x NB rats on blood pressure, plasma ouabain-like and marinobufagenin-like immunoreactivity, and on the activity of Na,K-ATPase in aortic sarcolemma were studied. The ACTH treatment for 8 days resulted in increased systolic blood pressure (151 +/- 12.4 v 121 +/- 4.0 mm Hg, P < .01), inhibition of Na,K-ATPase in aortic sarcolemma (2.99 +/- 0.35 v 5.43 +/- 0.17 micromol ADP/mg(prot)/h), and increases in plasma concentration of marinobufagenin-like (0.44 +/- 0.06 v 0.21 +/- 0.05 nmol/L), but not ouabain-like (0.09 +/- 0.01 v 0.10 +/- 0.04 nmol/L) immunoreactivity. In dissociation enhanced lanthanide fluoroimmunoassay (DELFIA), serial dilutions of plasma from ACTH-treated rats extracted with 25% and 80% acetonitrile, respectively, demonstrated parallelism to the calibration curves of ouabain and marinobufagenin. These findings suggest that an endogenous bufodienolide Na,K-ATPase inhibitor, rather than an endogenous ouabain-like compound, is increased after 8 days of treatment of rats with ACTH.

Adrenocorticotropic Hormone↗