Matrix metalloproteinase-2 and -9 in the granulomatous fibrosis of rats infected with Angiostrongylus cantonensis.

The histomorphology of granuloma formation and gelatinase production were investigated in the brains, hearts, lungs and livers of Sprague-Dawley rats infected with Angiostrongylus cantonensis. The relationships between two gelatinases and granulomatous fibrosis were explored, following infection of each rat with 60 infective larvae of the nematode. Worm recovery from the brain was maximal on day 15 post-inoculation whereas peak recovery from the lungs was maximal 75 days later, on day 90. The granulomatous reactions and fibrosis were marked in the lungs but only mild, if present at all, in the brain, heart and liver. Gelatin zymography revealed that matrix metalloproteinase2 (MMP-2) was present, at all time-points, in the heart and lungs, although only in the lungs was there partial conversion of the 72-kDa pro-enzyme to the 64-kDa active form during granulomatous fibrosis. The activity of the MMP-9 pro-enzyme was significantly higher at the time-points when granuloma formation was observed than at other times. Immuno-histochemistry revealed MMP-2 and MMP-9 within the lung granulomas, around infiltrating leucocytes and the epithelial cells of the alveoli. As the granulomatous fibrosis appeared to be strongly associated with MMP-2 and MMP-9, these enzymes may be useful markers in the lungs of rats infected with A. cantonensis.

Effects of tannic acid and its related compounds on food mutagens or hydrogen peroxide-induced DNA strands breaks in human lymphocytes.

The effect of tannic acid (TA), gallic acid (GA), propyl gallate (PA) and ellagic acid (EA) on DNA damage in human lymphocytes induced by food mutagens [3-amino-1-methyl-5H-pyrido (4,3-b) indole (Trp-P-2) and 2-amino-1-methyl-6-phenylimadazo (4,5-b) pyridine (PhIP) or H2O2 was evaluated by using single-cell electrophoresis (comet assay). The toxicity of these tested compounds (0.1-100 microg/ml) on lymphocytes was not found. These compounds did not cause DNA strand breaks at lower concentrations of 0.1-10 microg/ml. At a concentration of 100 microg/ml, TA and GA exhibited slight DNA damage, whereas PA and EA showed no DNA strand breaks. TA and its related compounds decreased the DNA strand breaks induced by Trp-P-2, PhIP or H2O2 at concentrations of 0.1-10 microg/ml. DNA repair enzymes endonuclease III (Endo III) and formamidopyrimidine-DNA glycoslase (FPG)] were used to examine the levels of oxidised pyrimidines and purines in human lymphocytes induced by H2O2. All the compounds at 10 microg/ml can reduce the level of FPG sensitive sites. However, only EA inhibited the formation of EndoIII sensitive sites. The results indicated that these compounds can enhance lymphocytes resistance towards DNA strand breaks induced by food mutagens or H2O2 in vitro.

Human Ca2+/calmodulin-dependent protein kinase kinase beta gene encodes multiple isoforms that display distinct kinase activity.

Ca(+2)/calmodulin-dependent protein kinases (CaMKs) are activated upon binding of Ca(+2)/calmodulin. To gain maximal activity, CaMK I and CaMK IV can be further phosphorylated by an upstream kinase, CaMK kinase (CaMKK). We previously isolated cDNA clones encoding human CaMKK beta isoforms that are heterogeneous in their 3'-sequences (Hsu, L.-S., Tsou, A.-P., Chi, C.-W., Lee, C.-H., and Chen, J.-Y. (1998) J. Biomed. Sci. 5, 141-149). In the present study, we examined the genomic organization and transcription of the human CaMKK beta gene. The human CaMKK beta locus spans more than 40 kilobase pairs and maps to chromosome 12q24.2. It is organized into 18 exons and 17 introns that are flanked by typical splice donor and acceptor sequences. Two major species of transcripts, namely the beta1 (5.6 kilobase pairs) and beta2 (2.9 kilobase pairs), are generated through differential usage of polyadenylation sites located in the last and penultimate exons. Additional forms of CaMKK beta transcripts were also identified that resulted from alternative splicing of the internal exons 14 and/or 16. These isoforms display differential expression patterns in human tissues and tumor-derived cell lines. They also exhibit a distinct ability to undergo autophosphorylation and to phosphorylate the downstream kinases CaMK I and CaMK IV. The differential expression of CaMKK beta isoforms with distinct activity further suggests the complexity of the regulation of the CaMKK/CaMK cascade and an important role for CaMKK in the action of Ca(+2)-mediated cellular responses.

Cloning, expression and chromosomal localization of human Ca2+/calmodulin-dependent protein kinase kinase.

A human cDNA clone encoding the calcium/calmodulin-dependent protein kinase kinase (CaMKK) was isolated by RT-PCR amplification of the fragment corresponding to the conserved kinase catalytic domain followed by rapid amplification of cDNA ends and cDNA library screening. Compilation of nucleotide sequencing data yielded a consensus cDNA sequence of 1.9 kb with an open reading frame of 1,251 nucleotides in length which translates to a polypeptide of 417 amino acids (47 kd). It showed significant homology to the rat brain CaMKK isozymes. The human CaMKK, which was expressed as a Flag-tagged protein in human non-small cell lung cancer H- 1299 cells followed by immunoprecipitation with anti-Flag antibody, was shown to phosphorylate recombinant human CaMK I in a calcium/CaM-dependent fashion. Northern blot analysis revealed that human CaMKK is ubiquitously expressed, with brain showing the highest level of expression. The CaMKK gene is localized to human chromosome 12. The presence of cDNA clones with divergent 3' terminal sequences suggests a family of CaMKK variants which may arise from alternative splicing.

High frequency of rearrangement of the RET protooncogene (RET/PTC) in Chinese papillary thyroid carcinomas.

The activation of RET protooncogene, through chromosomal translocation, is unique to papillary thyroid carcinomas. Rearrangement of the RET kinase domain to 3 partner genes has been described, of which the RET/PTC1 is the most common. To investigate the frequency of RET rearrangement in Chinese papillary thyroid carcinomas, we have performed RT-PCR to amplify specific RET/PTC transcripts. Among the papillary thyroid carcinomas of 11 patients examined, we have identified 2 containing RET/PTC1, 3 containing RET/PTC2, and 1 containing RET/PTC3 oncogenes. Although the cause of the high frequency of RET/PTC oncogenes in Chinese papillary thyroid carcinomas is unknown, our study suggests that RET rearrangement is an important genetic lesion underlying the development of thyroid papillary carcinoma in Taiwan.

Evidence for a Na+/Ca2+ exchanger in neuroblastoma x glioma hybrid NG108-15 cells.

To determine whether NG108-15 cells contain a functional Na+/Ca2+ exchanger, we isotonically replaced extracellular Na+ with N-methyl-D-glucamine (NMG) and measured the effect on cytosolic Ca2+ concentration ([Ca2+]i) using the fluorescent Ca2+ indicator fura 2. Replacement with NMG alone had no effect on basal [Ca2+]i or the rise in [Ca2+]i evoked by 80 mM K+ or 10 microM bradykinin, but caused a larger [Ca2+]i increase when thapsigargin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) were added to the cells; this enhanced [Ca2+]i increase could be reversed by adding Na+ back to the bathing buffer. The elevation in [Ca2+]i induced by thapsigargin and FCCP was inversely proportional to extracellular Na+ concentration. Furthermore, the exchanger operated in the reverse mode, as measured by either [Ca2+]i change or 45Ca2+ uptake. An 810 bp cDNA fragment of the exchanger was amplified by PCR; it differed by a single amino acid residue from the corresponding segment of the rat brain Na+/Ca2+ exchanger. These data suggest that a functioning Na+/Ca2+ exchanger exists in NG108-15 cells.

Two distinct ATP signaling mechanisms in differentiated neuroblastoma x glioma hybrid NG108-15 cells.

The ATP signaling mechanism in neuroblastoma x glioma hybrid NG108-15 cells differentiated by exposure to dibutyryl-cAMP was characterized. In cells loaded with fura-2, ATP rapidly raised the cytosolic Ca2+ concentration ([Ca2+]i); the magnitude of the rise was inversely proportional to the extracellular Na+ concentration. Large increases in cytosolic Na+ concentration, measured with the fluorescent Na+ indicator sodium-binding benzofuran isophthalate, were dose-dependently elicited by ATP. ATP also evoked the entry of ethidium bromide into cells, and this process was inhibited by Mg2+. Inositol-1,4,5-trisphosphate (IP3) generation induced by ATP was totally blocked by removal of extracellular Ca2+, but residual IP3 generation still remained in nondifferentiated cells. In addition, ATP produced a concentration-, time-, and Mg(2+)-dependent biphasic uptake of 45Ca2+. A range of nucleotides and ATP analogues, including CTP, UTP, and GTP, induced only 9-29% of the ATP response. However, adenosine 5'-thiotriphosphate evoked 79% of ATP-induced 45Ca2+ uptake. 45Ca2+ uptake elicited by ATP could be potently blocked by purinoceptor antagonists, but other tested reagents less effectively blocked the action of ATP. When bradykinin was used as an agonist, the [Ca2+]i rise was transient and was insensitive to the extracellular Na+ concentration. Na+ influx, entry of ethidium bromide, and 45Ca2+ uptake were unaffected by bradykinin. Furthermore, bradykinin-evoked IP3 generation was insensitive to extracellular Ca2+. Neither ATP nor bradykinin had any effect on cAMP levels within cells. These data suggest that ATP induces a [Ca2+]i rise in differentiated NG108-15 cells via two distinct Ca2+ influx mechanisms, i.e., a receptor-operated cation channel and pores formed by ATP4-. These mechanisms are distinct from those elicited by bradykinin.

The ClusNet algorithm and time series prediction.

This paper describes a novel neural network architecture named ClusNet. This network is designed to study the trade-offs between the simplicity of instance-based methods and the accuracy of the more computational intensive learning methods. The features that make this network different from existing learning algorithms are outlined. A simple proof of convergence of the ClusNet algorithm is given. Experimental results showing the convergence of the algorithm on a specific problem is also presented. In this paper, ClusNet is applied to predict the temporal continuation of the Mackey-Glass chaotic time series. A comparison between the results obtained with ClusNet and other neural network algorithms is made. For example, ClusNet requires one-tenth the computing resources of the instance-based local linear method for this application while achieving comparable accuracy in this task. The sensitivity of ClusNet prediction accuracies on specific clustering algorithms is examined for an application. The simplicity and fast convergence of ClusNet makes it ideal as a rapid prototyping tool for applications where on-line learning is required.

Real-time monitoring of crystallization and structural transformation of titania films with Raman spectroscopy.

The feasibility of using Raman spectroscopy to monitor the process of crystallization and structural transformation of thin films in real time has been demonstrated experimentally. Although only amorphous titania films are investigated, the technique can be applied to other optical films and to probe the deposition of such films as well.

The kinetics of uptake of 5-fluorouracil by rat liver.

1 Slices of rat liver weighing 15-30 mg were incubated at 37 degrees C in 5-fluorourecil (5-FU) solution. 2 The 5-FU taken up was measured by high pressure liquid chromatography. 3 Uptake of 5-FU was found to approximate to Michaelis-Menton kinetics with the values of Kt and Vmax equal to 15.39 mmol 1-1 and 1.96 mumol g-1 min-1 respectively. 4 Transport of 5-FU was saturable with the uptake being much greater in the non-fasting rats. 5 Intracellular concentration of 5-FU after 24 min incubation approached but did not significantly exceed that in the extracellular fluid. 6 Enzymatic destruction of 5-FU at 2 min was negligible but substantial after a long incubation period. 7 2,4-Dinitrophenol was found to inhibit transport of 5-FU significantly. 8 Results suggested that the mechanism of uptake of 5-FU by rat liver is an energy requiring process.

Determination of 5-fluorouracil in human plasma by high-pressure ion-exchange chromatography.

A method has been developed for the measurement of 5-fluorouracil by isocratic high-pressure ion-exchange liquid chromatography. Chlorpheniramine was added to the standards and samples before extraction with 16% isopropanol in ethyl acetate. The extracts were then chromatographed using a mobile phase of 0.014 mol/l ammonium dihydrogen phosphate buffer, pH 3.5, at a flow rate of 1 ml/min. Ultraviolet detection at 266 nm was used, and quantitation was by measurement of the peak height ratios of 5-fluorouracil to chlorpheniramine.

Volume changes induced in rabbit polymorphonuclear leukocytes by chemotactic factor and cytochalasin B.

Incubation of rabbit neutrophils with various chemotactic factors causes an expansion of their volume. The expansion shows a high correlation with the chemotactic responsiveness of these cells, requires metabolic energy, and is independent of the presence of divalent cations. Cytochalasin B causes a decrease in the volume of the neutrophil. This decrease also requires metabolic energy and is independent of divalent cations. In the presence of cytochalasin B, the chemotactic factor, instead of acting to expand cell volume, induces a further contraction of the cell; this decrease requires Ca2+ in the external medium.