Differential gene expression of chloroplast and cytosolic phosphoglycerate kinase in tobacco.

Northern blot analysis of RNA extracted from leaves of increasing age and different organs, indicates that genes encoding both isoenzymes of tobacco phosphoglycerate kinase (PGK, EC 2.7.2.3) are differentially expressed in a developmental and tissue-specific manner. The genes for both chloroplast PGK (chl-PGK) and cytosolic PGK (cyt-PGK) also show light-modulated gene expression in vivo. In dark-grown developing cotyledonary leaves of tobacco both PGK mRNAs are present, but only the concentration of the chl-PGK mRNA increased on illumination. In contrast, on transfer to darkness, the concentration of both mRNAs decreased in light-grown seedlings and then increased again on resumption of illumination.

Assessment of breast cancer size: a comparison of methods.

Two hundred women presenting with primary breast carcinoma were studied to find the most accurate single or combination of methods to assess breast tumour size. Correlations of the maximum clinical, mammographic and ultrasound tumour diameter were made with maximum histological diameter. Tumour size could be assessed clinically in all 200 patients, and overestimated the size of small tumours and underestimated large tumours (P less than 0.001). Mammographic measurement, which was possible in 145 (72.5%), underestimated the size of large tumours (P less than 0.01). Only 100 women underwent ultrasound examination (size assessed in 86%) and this modality tended to underestimate the size of all tumours (P less than 0.05). All methods of measurement showed similar correlations with histological size. Stepwise linear regression showed that the most accurate and practical estimation could be made using the formula: Histological size = 0.5 x mammographic size + 0.5 x clinical size. We conclude that clinical measurement of breast cancer size is as accurate as that from mammography or ultrasound. Accuracy can be improved by the use of a simple formula of both clinical and mammographic measurements.

The development of the activity of the chloroplastic and cytosolic isoenzymes of phosphoglycerate kinase during barley leaf ontogenesis.

The catalytic activities of the chloroplastic and cytosolic isoenzymes of phosphoglycerate kinase (PGK; EC 2.7.2.3) have been followed during the development of the first leaf of barley (Hordeum vulgare L.) grown for 7 d in darkness followed by transfer to continuous illumination. The investigation has included both the study of a standard leaf section, measured from the leaf tip, over the whole life of the leaf and the study of serial sections of leaf, measured from the leaf base, at a standard sampling time. The results of both approaches were fully compatible. As the catalytic activity of each isoenzyme in the standard assay is directly proportional to the amount of isoenzyme protein present, the catalytic activities may be interpreted wholly in terms of enzyme synthesis and degradation. Both isoenzymes are synthesized in darkness and in etiolated barley are present at a ratio of about 26∶74 for the cytosolic to chloroplastic isoenzymes. Illumination results in a fivefold or greater increase in chloroplast PGK over a number of days with little change of the cytosolic isoenzyme, resulting in an eventual ratio of cytosolic to chloroplastic isoenzymes approaching the green-leaf value of about 9∶91. Prior to any detectable onset of senescence a 15-fold increase in cytosolic isoenzyme commenced while the amount of chloroplast PGK remained constant. It is suggested that the increased cytosolic PGK may be involved in the export of carbohydrate reserves (starch) prior to leaf senescence. Both isoenzymes subsequently decline in parallel to total protein and chlorophyll in the course of senescence.

Pancreaticogastrostomy after pancreaticoduodenectomy.

Although the mortality following pancreaticoduodenectomy has fallen and is now below 5%, overall 14% of patients develop a leak at the pancreatic anastomosis. This complication carries a 24% mortality rate when pancreaticojejunostomy is the method of reconstruction. In order to reduce the incidence of this complication, pancreaticogastrostomy can be performed following pancreaticoduodenectomy. A total of 41 patients underwent this operation between 1968 and 1989. The indications for operations were carcinoma of the head of the pancreas (n = 19), carcinoma of the ampulla (n = 12), carcinoma of the lower end of the common bile duct (n = 6), chronic pancreatitis (n = 3) and one patient with a nonfunctioning islet cell tumour. One patient developed a pancreatic fistula which closed spontaneously in 5 days. This patient is alive and well 36 months after operation. Pancreaticogastrostomy with pancreatic duct to gastric mucosa anastomosis is recommended as a safe and straight-forward method of reconstruction following pancreaticoduodenectomy.

Combined tamoxifen and anabolic steroid as primary treatment for breast carcinoma in the elderly.

Fifty-one consecutive patients aged over 70 years with localized breast carcinoma received primary treatment with tamoxifen and nandrolone decanoate. Results were compared to a control group of 51 age, tumour volume and nodal status-matched patients selected from a cohort who had primary treatment with tamoxifen alone. One year after commencement of treatment 31 (60%) patients treated with tamoxifen and nandrolone showed regression of their tumour, compared to 24 (47%) in the control group. Twenty-two (44%) patients had side effects attributable to nandrolone. Only four (8%) patients continued nandrolone for more than 12 months, and at 3 years after commencement of treatment 21 (40%) patients from each group had tumour regression. We argue that nandrolone should not be used as primary treatment in breast carcinoma, but may have a role to play in patients who fail to respond to tamoxifen.

Separation, purification, and comparative properties of chloroplast and cytoplasmic phosphoglycerate kinase from barley leaves.

The chloroplast and cytoplasmic isoenzymes of phosphoglycerate kinase (PGK) (EC. 2.7.2.3) from Hordeum vulgare leaves have been separated and purified for the first time to apparent homogeneity. The method for purifying the isoenzymes is described here and consists of DEAE Sephacel chromatography followed by affinity chromatography on ATP Sepharose. This consistently provided a 500- to 900-fold purification of each isoenzyme. Most of the total PGK in green barley leaves was found to be in the chloroplasts with only 10% in the cytoplasm. The immunological properties of the two isoenzymes were compared. The antisera raised to the separate isoenzymes showed cross-reactivity, although there is evidence that each isoenzyme possesses some distinct epitopes. The isoenzymes differ in overall charge with isoelectric points at 5.2 and 5.4 for the chloroplast and cytoplasmic isoenzymes, respectively. Molecular mass estimations by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis provided similar values of approximately 38 kilodaltons for each isoenzyme, some 4 to 5 kilodaltons less than the values calculated from the cDNA sequences of the wheat isoenzymes. The isoenzymes have broadly similar pH optima of pH 7 to 8. The cytoplasmic isoenzyme is more thermally stable than the chloroplast isoenzyme. Further studies are now in progress to compare both the regulatory properties of the isoenzymes and also their three-dimensional structures as compared with the yeast enzyme.

Wheat phosphoglycerate kinase: evidence for recombination between the genes for the chloroplastic and cytosolic enzymes.

We have isolated and sequenced cDNA clones containing the entire coding region of both the chloroplast and cytosolic versions of phosphoglycerate kinase from wheat. Comparison of these sequences reveals a higher than expected level of similarity between the nucleic acids and encoded proteins. Analysis of this data in relation to that for phosphoglycerate kinase sequences of mammals, prokaryotes and yeasts suggests that the wheat genes have recombined. This has resulted in the chloroplast and cytosolic kinases being more similar to each other than would be expected if the chloroplast enzyme had evolved directly from that of a prokaryotic progenitor.

Out-patient diagnosis of breast cancer.

A method of diagnosis of the solid discrete breast lump using frozen section examination of a Tru-cut needle biopsy is described. In 194 patients, the Tru-cut biopsy report is compared to the histological report of the excised lesion. Overall accuracy is 90 per cent, sensitivity 89 per cent, specificity 96 per cent, predictive value of a positive report 99 per cent, and predictive value of a negative report 56 per cent. The advantage of the technique is that many patients with breast cancer can be informed of the diagnosis at their first attendance.

Proline and glycine betaine influence protein solvation.

Glutamine synthetase from barley (Hordeum distichum L.) is precipitated by polyethylene glycol (PEG). Proline, in a concentration-dependent manner, reduces the amount of enzyme precipitated by PEG, although the effect of the imino acid can be counteracted by raising the level of PEG. The effect of PEG is a function of mer number and concentration and the influence of both elements can be ameliorated by proline. PEG-induced enzyme precipitation is a function of pH, as is its interaction with both proline and betaine in the reaction. The lack of effect of amount of enzyme on the proline and PEG effects supports the conclusion that, in this system, proline and PEG do not function through interaction with the protein. Other compounds, such as glycine, glucose, and sucrose, can decrease the PEG-induced precipitation of the enzyme, although glycerol was not active under the conditions employed.The results are consistent with the proposition that a protein-containing system in which high concentrations of proline and/or betaine are present, is better ;protected' against the biologically unfavorable consequences of dehydration-induced thermodynamic perturbation.

Primary treatment of breast cancer in elderly women with Tamoxifen.

One hundred and sixty-one female patients, over the age of 70, with breast cancer were treated by Tamoxifen alone in a pilot phase II study. 61% showed objective regression of the disease and in 27% there was complete remission. The treatment was well tolerated and the survival rate compared favourably to that following conventional methods of treatment. An accurate assessment of the method awaits a randomized clinical trial.

The presentation and progress of macroscopic breast cysts.

A total of 322 cases of macroscopic breast cysts presenting at a district general hospital has been studied with a minimum follow-up period of 5 years. In almost half the patients (44.18 per cent) a further cyst occurred following aspiration, and in two-thirds of these, more than one subsequent cyst developed. There was a 50-year age span from the youngest to the oldest patient at presentation: however, the majority of cysts (61.80 per cent) occurred in the perimenopausal age group (40-50 years). The subsequent incidence of carcinoma was more than twice that which would be expected in the population at large, but no case of carcinoma that was not suspected clinically was revealed by cytology.

Activation of Glyceraldehyde-Phosphate Dehydrogenase (NADP) and Phosphoribulokinase in Phaseolus vulgaris Leaf Extracts Involves the Dissociation of Oligomers.

Phosphoribulokinase (EC 2.7.1.19, ATP: d-ribulose-5-phosphate-1-phosphotransferase) resembles the NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13, d-glyceraldehyde-3-phosphate: NADPH(+) oxidoreductase [phosphorylating]) of chloroplasts in that the activation of both of these enzymes involves the dissociation of oligomers (apparently tetrameric forms) with low catalytic activity to give protomers which possess higher catalytic activity. Gel filtration on Sepharose 6B has shown that the molecular weights of the oligomer and active protomer of phosphoribulokinase are, respectively, about 6.8 x 10(5) and 1.7 x 10(5), whereas the corresponding values for glyceraldehyde-3-phosphate dehydrogenase are 8.2 x 10(5) and 2.2 x 10(5). Activation of both enzymes occurs in response to either ATP, dithiothreitol, or cholate while the glyceraldehyde-3-phosphate dehydrogenase is also activated by NADPH. Activation/dissociation of these enzymes may involve conformational changes resulting from nucleotide binding, the reduction of sulfur bridges, and the cholate induced loosening of hydrophobic interactions.

Studies in seed dormancy : IX. The role of gibberellin biosynthesis and the release of bound gibberellin in the post-chilling accumulation of gibberelin in seeds of Corylus avellana L.

When dormant hazel seeds were subjected to six weeks chilling at 5° C their subsequent transfer to 20° C resulted in the accumulation of gibberellin (GA) followed by germination. In the presence of either phosphon D or ß-chlorethyltrimethylammonium chloride (CCC) at 20° C there was inhibition of both GA accumulation and germination, a finding consistent with the hypothesis that GA biosynthesis is a necessary prerequisite for the germination of chilled hazel seeds. As abscisic acid showed a strong inhibition of germination but had little effect on GA accumulation it is presumed not to have affected GA biosynthesis but to have inhibited GA action. These conclusions were supported by experiments in which the interaction of exogenous GA3 with growth retardants and ABA was tested on the germination of chilled hazel seeds. Experiments in which the embryonic axes and cotyledons of chilled seeds were incubated separately at 20° C established that GA biosynthesis de novo occurred in the embryonic axis and indicated that in the intact seed some of the GA would have been translocated to the cotyledons. The isolated cytoledons showed no GA biosynthesis de novo but gave some release of GA from one or more bound forms.

Plastid development in primary leaves of Phaseolus vulgaris. Development of plastid adenosine triphosphatase activity during greening.

The etioplasts of dark-grown bean leaves showed ATPase (adenosine triphosphatase) activity which had a pH optimum of 8.5, was stimulated by dithiothreitol and unaffected by light-triggering. Bean chloroplasts showed a low activity of dark-induced ATPase with a pH optimum of 8.5 and a substantial amount of light-triggered activity with a pH optimum of 8.0. The light-triggered activity depended on dithiothreitol and Mg2+ and was promoted by phenazine methosulphate. Light-triggered ATPase activity was completely inhibited by 20mum-dicyclohexylcarbodi-imide. Etioplasts developed light-triggered ATPase activity in response to 30 min illumination of the etiolated leaves. During the 48 h of light-induced greening of dark-grown leaves there was a 70% increase of the chloroplast ATPase activity found after light-triggering and a 30% fall in the dark-induced activity, both expressed on a per leaf basis. As the larger part of these changes occurred during the first 30 min of illumination, it is concluded that most or all of the chloroplast ATPase was present in the etioplast, a conclusion identical with that of Lockshin et al. (1971) for maize. During 48 h of greening there was a tenfold increase in the amount of thylakoid membrane in the leaf together with an 83% fall in the ATPase activity per m2 of thylakoid membrane, measured after light-triggering.

Chloramphenicol as an energy transfer inhibitor in spinach chloroplasts.

At concentrations of up to 300 mug/ml both d-threo- and l-threo-chloramphenicol act as energy transfer inhibitors in spinach chloroplasts, in that they inhibit both phosphorylation and phosphorylating electron transport, without affecting the nonphosphorylating electron transport which occurs either in the absence of a phosphate acceptor or in the presence of the uncoupler ammonium chloride. At higher concentrations, there appears to be an additional site of chloramphenicol inhibition of electron transport. If d-threo-chloramphenicol is to be used as a protein synthesis inhibitor in intact chloroplasts or tissues, control experiments with another chloramphenicol isomer seem to be necessary.

Studies in seed dormancy : VIII. The identification and determination of gibberellins A1 and A 9 in seeds of Corylus avellana L.

The three major fractions (II, III and V) of the five fractions of gibberellin (GA)-like activity obtained by the thin layer chromatographic purification of an extract of hazel embryos have been examined by gas liquid chromatography and combined gas chromatography-mass spectrometry. By these techniques GA1 was identified as the methyl ester trimethylsilyl ether in fraction II and GA9 as the methyl ester in fraction V. Both substances were clearly separated from other components by gas liquid chromatography so that accurate determination of their concentrations was possible. GA1 accounted for most of the activity found by bioassay in fraction II while GA9 accounted for only part of the activity of fraction V. The GA-like substances in the less biologically active thin layer chromatography fractions have not yet been identified.Freshly harvested seeds contained GA1 but GA9 was not detected. After 28 days dry storage and 42 days imbibition and chilling at 5°, the GA1 content was very low and GA9 was still below the limits of detection. On subsequent exposure of the seeds to a temperature of 20° for 8 days, there was a 40-fold increase of GA1 and at least a 300-fold increase of GA9.

Plastid development in primary leaves of Phaseolus vulgaris: The light-induced development of the chloroplast cytochromes.

Etioplasts obtained from the primary leaves of dark-grown bean plants contained cytochromes f, b-559LP and b-563 in a molar ratio of approximately 1.0:2.0:1.5. On illumination of the plants there was a lag of between 10 and 15 h before these cytochromes increased in amount, but after 48 h they had increased from 6- to 10-fold on a per plastid basis. The presence of cytochrome b-559HP in the plastids was first detected after 15 h of illumination, which coincided with the commencement of grana formation and the onset of a number of photosynthetic reactions in the greening leaves. After 48 h of illumination the molar ratio for cytochromes f, b-559HP, b-559LP and b-563 was 1.0:1.2:2.8:2.6.Agranal chloroplasts formed by the exposure of dark-grown plants to intense light flashes contained high amounts of cytochromes f, b-559LP and b-563 but cytochrome b-559HP could not be detected.As the light-induced formation of cytochromes f, b-559LP and b-563 was substantially inhibited by D-threo chloramphenicol, but not by the L-threo isomer, it seems likely that their formation was dependent on 70S ribosomes. Both chloramphenicol isomers gave plastids which lacked cytochrome b-559HP.

Studies in seed dormancy : VII. The abscisic acid content of the seeds and fruits of Corylus avellana L.

ABA has been identified by GLC-MS and routinely determined by GLC as one of several inhibitory substances in the testa and pericarp of hazel nuts. Its concentration in newly harvested nuts, which had not developed embryo dormancy, was 19.0 nmoles/g dry weight for the testa, 1.4 nmoles/g for the pericarp and 0.09 nmoles/g for the embryo. Dry storage of the nuts resulted in the development of embryo dormancy together with a slight loss of ABA. On imbibition of dormant nuts at 5° C and 20° C there was a 61% loss of ABA from the testa and pericarp in both cases. However the 5° C imbibition resulted in the breaking of seed dormancy while the 20° C imbibition had no effect on the dormancy. The ABA of the testa and pericarp seems to be concerned with the maintenance of seed dormancy prior to the onset of embryo dormancy. Subsequent to the onset of embryo dormancy, ABA seems to show little effect on either the maintenance or breaking of seed dormancy.