Movement of newly assimilated 13C carbon in the grass Lolium perenne and its incorporation into rhizosphere microbial DNA.

One of the key processes that drives rhizosphere microbial activity is the exudation of soluble organic carbon (C) by plant roots. We describe an experiment designed to determine the impact of defoliation on the partitioning and movement of C in grass (Lolium perenne L.), soil and grass-sterile sand microcosms, using a (13)CO(2) pulse-labelling method. The pulse-derived (13)C in the shoots declined over time, but that of the roots remained stable throughout the experiment. There were peaks in the atom% (13)C of rhizosphere CO(2) in the first few hours after labelling probably due to root respiration, and again at around 100 h. The second peak was only seen in the soil microcosms and not in those with sterilised sand as the growth medium, indicating possible microbial activity. Incorporation of the (13)C label into the microbial biomass increased at 100 h when incorporation into replicating cells, as indicated by the amounts of the label in the microbial DNA, started to increase. These results indicate that the rhizosphere environment is conducive to bacterial growth and replication. The results also show that defoliation had no impact on the pattern of movement of (13)C from plant roots into the microbial population in the rhizosphere.

Comparison of the novel quantitative ARMS assay and an enriched PCR-ASO assay for K-ras mutations with conventional cytology on endobiliary brush cytology from 312 consecutive extrahepatic biliary stenoses.

BACKGROUND: Extrahepatic biliary stenosis (EBS) has malignant and benign causes. Patients with EBS are at risk of having or developing malignancy. Accurate diagnostic tests for early detection and surveillance are needed. The sensitivity of biliary cytology for malignancy is low. K-ras mutation analysis on brush cytology is a valuable adjunct, but specificity is low. A quantitative test for K-ras mutations has been developed: the amplification refractory mutation system (ARMS). AIM: To assess the test characteristics and additional value of ARMS in diagnosing the cause of EBS. METHODS: Brush samples from endoscopic retrograde cholangiopancreatography were collected from 312 patients with EBS. K-ras mutation analysis was performed using ARMS-allele specific amplification was coupled with real time fluorescent detection of PCR products. Results were compared with conventional cytology and K-ras mutation analysis using allele specific oligonucleotide (ASO) hybridisation, and evaluated in view of the final diagnosis. RESULTS: The test characteristics of ARMS and ASO largely agreed. Sensitivity for detecting malignancy was 49% and 42%, specificity 93% and 88%, and positive predictive value (PPV) 96% and 91%, respectively. The sensitivity of ARMS and cytology combined was 71%, and PPV was 93%. The specificity of ARMS could be increased to 100% by setting limits for the false positives, but reduced sensitivity from 49% to 43%. CONCLUSIONS: ARMS can be considered supplementary to conventional cytology, and comparable to ASO in diagnosing malignant EBS. A specificity of 100% can be achieved with ARMS, which should be considered in the surveillance of patients at risk for pancreatic cancer.

K-ras point mutation detection in lung cancer: comparison of two approaches to somatic mutation detection using ARMS allele-specific amplification.

BACKGROUND: The use of sensitive molecular techniques to detect rare cells in a population is of increasing interest to the molecular pathologist, but detection limits often are poorly defined in any given molecular assay. We combined the approaches of real-time quantitative PCR with ARMS(TM) allele-specific amplification in a novel assay for detecting mutant K-ras sequences in clinical samples. METHODS: ARMS reactions were used to detect seven commonly occurring mutations in the K-ras oncogene. These mutations produce amino acid changes in codon 12 (Gly to Ala, Arg, Asp, Cys, Ser, or Val) and codon 13 (Gly to Asp). A control reaction was used to measure the total amount of amplifiable K-ras sequence in a sample so that the ratio of mutant to wild-type sequence could be measured. Quantitative data were confirmed for a selection of samples by an independent cloning and sequencing method. The assay was used to analyze 82 lung tumor DNA samples. RESULTS: The assay detected K-ras mutations in 44% of adenocarcinomas, which is equivalent to frequencies reported in the literature using ultrasensitive techniques. Forty-six percent of squamous carcinomas were also positive. The ratio of mutant sequence in the tumor DNA samples was 0.04-100%. CONCLUSIONS: The assay is homogeneous, with addition of tumor DNA sample being the only step before results are generated. The quantitative nature of the assay can potentially be used to define the analytical sensitivity necessary for any specified diagnostic application of K-ras (or other) point mutation detection.

Interactions between the oestrogen and insulin-like growth factor signalling pathways in the control of breast epithelial cell proliferation.

There is increasing evidence for interactions between steroid and growth factor signalling pathways. Oestrogens modulate the responsiveness of breast epithelial cells to insulin-like growth factors (IGFs), and this may be the mechanism by which oestrogens modulate cell proliferation. Oestrogens appear to act at several points in the IGF signal transduction pathway. Despite earlier studies suggesting that breast epithelial cells do not synthesize IGF-I, we have shown by PCR that IGF-I is expressed and that its expression is regulated by oestrogen. IGF-II is expressed at markedly higher levels than IGF-I and is also regulated by oestrogen, consistent with it being an oestrogen-regulated autocrine growth factor. Oestrogens regulate the expression of IGF binding proteins and the type I IGF receptor. The biological significance of oestrogen regulation of IGF binding protein expression is not clear. Experiments in which the type I IGF receptor has been constitutively overexpressed have suggested that oestrogen regulation of the receptor is not involved in mediating the effects of oestrogen on cell proliferation. Recent studies have started to assess the effects of oestrogen on the expression of components of the IGF signal transduction pathway, and have shown that the expression of insulin receptor substrate-1, the principal substrate for the tyrosine kinase of the type I IGF receptor, is regulated by oestradiol.

Insulin-like growth factors control the regulation of oestrogen and progesterone receptor expression by oestrogens.

Ligands for the type I insulin-like growth factor (IGF) receptor interact with oestrogens to control the proliferation of oestrogen responsive breast cancer cells. The aim of this study was to determine the involvement of ligands for the type I IGF receptor in the regulation of oestrogen receptor (OR) expression by oestrogens and antioestrogens in these cells. Oestrogen decreased OR mRNA levels in MCF-7 cells whereas it increased them in T47D, EFM-19 and ZR-75 cells. In MCF-7 cells, IGF-I and insulin lowered further OR expression in the presence of oestrogen. In the presence of IGF-I or insulin, the induction of progesterone receptor mRNA by oestradiol was considerably attenuated in MCF-7 cells, showing that the enhanced down-regulation of OR mRNA levels influenced the expression of oestrogen-regulated genes. The oestrogen agonist activity of the antioestrogens tamoxifen and ICI 182 780 for the down-regulation of OR expression in MCF-7 cells was modulated by type I IGF receptor ligands. Overall these experiments show that OR expression is differentially regulated by oestrogen in individual oestrogen-responsive breast cancer cell lines. Ligands for the type I IGF receptor can modulate regulation of OR expression by oestrogens and antioestrogens principally in cells in which oestrogens down-regulate OR expression.

Resolution of racemic carbocyclic analogues of purine nucleosides through the action of adenosine deaminase. Antiviral activity of the carbocyclic 2'-deoxyguanosine enantiomers.

The action of adenosine deaminase on racemic carbocyclic analogues of 6-aminopurine nucleosides was investigated. When either racemic carbocyclic adenosine [(+/-)-C-Ado] or the racemic carbocyclic analogue [(+/-)-C-2,6-DAP-2'-dR] of 2,6-diaminopurine 2'-deoxyribofuranoside was incubated with this enzyme, approximately half of the material was deaminated rapidly. From the resulting solution, the D isomers of the deaminated carbocyclic analogues (D-carbocyclic inosine, D-C-Ino, or D-carbocyclic 2'-deoxyguanosine, D-2'-CDG) and the L isomers of the undeaminated carbocyclic analogues were isolated. At higher concentrations of the enzyme, deamination of L-C-Ado and L-C-2,6-DAP-2'-dR proceeded slowly, thus also making the other enantiomers accessible. In tests in vitro against herpes simplex virus, types 1 and 2, D-2'-CDG was as active and potent as (+/-)-2'-CDG, whereas L-2'-CDG displayed only modest activity. In contrast to the previously reported high activity and potency of (+/-)-C-2,6-DAP-2'-dR against these two viruses, L-C-2,6-DAP-2'-dR was inactive.

(+/-)-3-(4-Amino-1H-pyrrolo[2,3-d]pyrimidin-1-yl)-5-(hydroxymethyl)- (1 alpha,2 alpha,3 beta,5 beta)-1,2-cyclopentanediol, the carbocyclic analogue of tubercidin.

(+/-)-3-(4-Amino-1H-pyrrolo[2,3-d]pyrimidin-1-yl)-5-(hydroxymethyl)- 1 alpha,2 alpha,3 beta,5 beta)-1,2-cyclopentanediol (9), the carbocyclic analogue of tubercidin, prepared from (+/-)-3-amino-5-(hydroxymethyl)-(1 alpha,2 alpha,3 beta,5 beta)- 1,2-cyclopentanediol (6), is cytotoxic to cells containing adenosine kinase but not to cells that do not, indicating that its activity depends on phosphorylation. Although inactive against P388 leukemia in mice and against herpes and influenza viruses in vitro, it showed marginal activity against respiratory syncytial, vesicular stomatitis, and rhino viruses in vitro.

The distribution of amyloid P component in normal human cervix.

The distribution of amyloid P component in normal human adult cervix was studied using fluorescent immunohistochemical techniques on frozen sections. Amyloid P component is associated with elastic fibres which are particularly concentrated in a sub-epithelial plexus in the ectocervix. This plexus does not extend into the endocervix but terminates at, or just caudal to, the squamocolumnar junction. Amyloid P component was not demonstrated in any of the epithelial basement membranes.

Carbocyclic analogue of 3-deazaadenosine: a novel antiviral agent using S-adenosylhomocysteine hydrolase as a pharmacological target.

The carbocyclic analogue of 3-deazaadenosine (3-deaza-C-Ado) has been synthesized and found to have antiviral activity in cell culture against herpes simplex virus type 1, vaccinia virus, and HL-23 C-type virus. It is relatively noncytotoxic at effective antiviral concentrations and is not subject to deamination or phosphorylation. It acts as a competitive inhibitor of S-adenosyl-L-homocysteine hydrolase, is at best a poor substrate, and does not inactivate the enzyme significantly. 3-Deaza-C-Ado may cause a selective inhibition of the methylation of the polynucleotide 5' cap of viral mRNA via higher cellular concentrations of S-adenosyl-L-homocysteine, resulting from the inhibition of S-adenosylhomocysteine hydrolase in infected cells, since increases in the intracellular level of S-adenosylhomocysteine, but no effects on DNA or RNA synthesis, were observed after incubation of these cells with it.

Nucleosides of 2-azapurines. 7 H-Imidazo[4,5-d]-1,2,3-triazines. 2.

A number of nucleosides of 2-azaadenine (4-amino-7H-imidazo[4,5-d]-1,2,3-triazine) were prepared by a previously described route, and some of these were deaminated by means of adenosine deaminase. Alternatively, nucleosides of 2-azahypoxanthine (7H-imidazo[4,5-d]-1,2,3-triazin-4(3h)-one) were prepared from hypoxanthine nucleosides by a 2-azahypoxanthine (7H imidazo[4,5]-1,2,3-triazin-4(3H)-one) were prepared from hypoxanthine nucleosides by a ring-opening and reclosure sequence. The cytotoxicity of these compounds to human epidermoid carcinoma No. 2 cells in culture and to certain resistant sublines thereof was determined. 2-Azaadenine nucleosides chan be metabolized to nucleotides, the cytotoxic agents, by two pathways, but the activity of the 2-azahypoxanthine nucleosides appears to result only from cleavage back to 2-azahypoxanthine.