Modifications to a Vitis Shoot Tip Cryopreservation Procedure: Effect of Shoot Tip Size and Use of Cryoplates.

BACKGROUND: Cryopreservation has been considered a preferred method for the long-term storage of plant germplasm, especially to efficiently conserve and maintain the genetic integrity of genebank materials. Droplet-vitrification (DV) procedures have been developed to cryopreserve Vitis shoot tips from in vitro-grown plants. OBJECTIVE: This research focused on optimizing shoot tip sizes for DV and the feasibility of using cryo-plates for Vitis cryopreservation. MATERIALS AND METHODS: Uniform shoot tips were obtained from nodal sections cultured from in vitro-grown stock plants of Vitis aestivalis and Vitis jacquemontii (PI 135726). Shoot tips were precultured for 3 days on medium containing 0.3 M sucrose, salicylic acid, glutathione (reduced form), and ascorbic acid. They were cryopreserved using either DV on aluminum foil strips or by placement in calcium alginate gel in the wells of aluminium cryo-plates (V cryo-plate method). Shoot tips were then treated with loading solution followed by PVS2 treatment prior to liquid nitrogen (LN) exposure. Shoot tips were warmed in unloading solution and placed on recovery medium. The effect of extraction or non-extraction of the cryopreserved shoot tips from alginate beads was also tested. RESULTS: The highest regrowth levels of cryopreserved shoot tips were obtained using 1 mm shoot tips and a PVS2 exposure for 90 min at 0°C with the DV method on aluminum foil strips or by using 30 min of PVS2 at 22°C using V cryo-plates. CONCLUSION: Shoot tip size is an important factor in the cryopreservability of Vitis shoot tips; 1 mm shoot tips were the most successful for the DV cryopreservation method that was tested. In addition, the V cryo-plate cryopreservation technique described herein can be easily executed and results in high regrowth levels (≥70%) with quality plants obtained from cryo-exposed shoot tips, making it a practical and promising Vitis cryopreservation methodology.

Successful Cryopreservation of Vitis Shoot Tips: Novel Pre-treatment Combinations Applied to Nine Species.

BACKGROUND: Secure back-up of Vitis genetic resource collections requires cryopreservation methods that give long-term survival of clonal germplasm having diverse genetic backgrounds. OBJECTIVE: This work sought to increase survival of Vitis shoot tips exposed to liquid nitrogen using combinations of pretreatments and cryoprotection procedures. The new procedure should give high survival of shoot tips from a wide range of Vitis species. MATERIALS AND METHODS: In vitro plants from nine Vitis species were used as source material for nodal sections. Shoot tips were then excised from nodal sections that were grown on medium containing benzyladenine, salicylic acid, glutathione, and ascorbic acid. The shoot tips were treated with loading solution, and then half-strength PVS2 for 30 minutes, prior to full-strength PVS2 treatments for between 60 and 90 minutes prior to liquid nitrogen (LN) exposure. RESULTS: Shoot tip regrowth levels were highest 90 minutes in PVS2+LN and ranged from 24-43% and averaged 35±2% across the nine Vitis species. CONCLUSION: The pretreatment, cryopreservation, and recovery methods yielded successful regrowth for multiple Vitis species using a droplet-vitrification procedure.

Hepatic iron in dialysed patients given intravenous iron dextran.

Five percutaneous biopsy and 17 necropsy liver specimens were analysed histologically and chemically for iron content in 22 patients receiving dialysis for chronic renal failure, 13 of whom were given intravenous iron-dextran. Brissot scores for assessing histological hepatic iron deposition and chemically measured liver iron concentrations correlated closely. Both variables depended on total cumulative dose of iron, and to a lesser extent, on time since the last dose. Fibrosis (seen in five patients) was minimal and non-specific. Electron microscopic examination showed that there was no generalised damage and confirmed the presence of iron in the hepatocytes in the form of ferritin. High liver iron concentrations, in excess of 1000 micrograms/100 mg dry weight, were seen in two patients. Four others given comparable cumulated amounts (18-23 g iron) did not have such high concentrations. Plasma ferritin concentrations were high in eight patients, some with and some without fibrosis. The risk of temporarily high iron deposition in the liver causing damage seemed to be minimal when weighed against the benefit of increased haemoglobin in most of the patients. Intravenous iron treatment merits further evaluation, particularly with the advent of erythropoietin treatment, which requires continuously available iron.

A novel means of comparing the bioequivalence of two formulations of tripotassium dicitrato bismuthate (De-Noltab).

Tripotassium dicitrato-bismuthate (TDB) has a topical action in the upper gastrointestinal tract. Absorption is minimal and the blood levels are therefore irrelevant to the assessment of bioavailability. TDB is however detectable in the stomach and duodenum by conventional electron microscopy. Following oral administration of two different formulations of TDB tables to patients attending a gastroscopy clinic, duodenal biopsies were obtained and examined by electron microscopy. The amount of electron dense drug precipitate was quantified. No difference could be detected between the two formulations.

The influence of ascites on the pharmacokinetics of piretanide in cirrhotic patients.

The pharmacokinetics of piretanide, a new loop diuretic, were studied in seven patients with severe liver disease before and after resolution of ascites. The time to maximum concentration was significantly prolonged by the presence of ascites. Tmax after relief of ascites was similar to that seen for normal volunteers. Area under the curves, bioavailability, volumes of distribution and elimination half-lives did not change after resolution of the ascites: two patients in whom diuretic resistant ascites occurred showed similar pharmacokinetics to that of the diuretic responders. Reduced responsiveness to piretanide therapy in patients with gross ascites does not appear to be the result of decreased bioavailability.

Effects of the angiotensin converting enzyme inhibitor, captopril, in essential hypertension.

1 The effects of the orally active angiotensin converting enzyme inhibitor, captopril, were examined in 15 patients with mild or moderate essential hypertension. 2 Following initial dosing with captopril 25 mg, there was a significant fall in supine and erect blood pressure in 2 h and lasting for 6 h. There was no significant alteration of heart rate. No reduction was seen in plasma noradrenaline concentration. Maximum inhibition of plasma converting enzyme activity occurred 60 min post-dosing. 3 There was a strong positive correlation between blood pressure reduction and converting enzyme inhibition. The magnitude of the blood pressure reduction and converting enzyme inhibition following initial dosing was reflected in subsequent success with captopril monotherapy during a 12 week follow-up period. 4 It was also found that patients who did not achieve adequate blood pressure control on captopril in doses of 50 mg three times daily or less still were not controlled on a dose of 150 mg three times daily without the addition of a diuretic. 5 The risks of renal and marrow toxicity from captopril may be reduced by administering only low doses with the addition, where necessary, of a thiazide diuretic.

Effects of captopril, an angiotensin-converting enzyme inhibitor, in normotensive sodium-replete volunteers.

Captopril (SQ 14 225) was administered to normotensive, sodium-replete volunteers in order to investigate the relationships between its haemodynamic effects and effects on sympathetic activity, the renin-angiotensin-aldosterone system, and plasma-converting enzyme activity. Following the administration of captopril (25 mg) orally, mean arterial pressure fell (supine 89.1 +/- 5.9 to 81.1 +/- 3.3 mm Hg, p less than 0.01; erect 98.5 +/- 5.5 to 87.4 +/- 6.7 mm Hg, p less than 0.01) but heart rate did not change. Plasma noradrenaline rose; plasma renin activity and angiotensin I concentration rose, while angiotensin II and aldosterone fell; plasma-converting enzyme was inhibited for 6 h. The extent of blood pressure reduction and converting enzyme inhibition was closely correlated (r = 0.92, p less than 0.001). Plasma captopril concentration was directly related to converting enzyme inhibition in an in vitro study using plasma from the same subjects. In the absence of a convenient assay of captopril, converting enzyme may be used as an index of captopril concentration in the further study of kinetic and dynamic relationships.

Pathological fracture, bone metastases and primary hepatocellular carcinoma.

Pathological fracture and metastatic bone disease without pulmonary metastases were the presenting features in three patients ultimately found to have primary hepatocellular carcinoma. In one case immunoperoxidase staining of bone scrapings for alpha-fetoprotein secreting cells contributed directly to the diagnosis.

The influence of captopril, the nitrates and propranolol on apparent liver blood flow.

Indocyanine green estimated apparent liver blood flow was measured in normal volunteers following glyceryl trinitrate, propranolol, isosorbide mononitrate and captopril. Glyceryl trinitrate and propranolol significantly reduced apparent liver blood flow. Isosorbide mononitrate did not alter apparent liver blood flow or produce an additional reduction in apparent liver blood flow when combined with propranolol. Captopril did not alter apparent liver blood flow despite a significant fall in mean arterial pressure and rise in plasma renin activity. Captopril and isosorbide mononitrate if shown to reduce portal pressure, do so without a fall in apparent liver blood flow.

Mefenamic acid-induced bullous pemphigoid.

A 52 year old man developed bullous pemphigoid, Coombs' positive haemolytic anaemia and diarrhoea related to the use of mefenamic acid. Clinical manifestation of the bullous pemphigoid, haemolytic anaemia and diarrhoea resolved following discontinuation of the mefenamic acid. Mefenamic acid should be added to the list of agents that are known to induce bullous pemphigoid.

Diagnosis of type 1a and type 1c glycogen storage diseases in adults.

The hepatic glucose-6-phosphatase system was studied with a novel microanalytical technique in adult patients undergoing liver biopsy. 4 patients were diagnosed as having type 1 glycogen storage disease (GSD). 3 of these patients, who had hypoglycaemic symptoms, had variations of type 1a GSD, which is caused by a defect in the hepatic microsomal glucose-6-phosphatase enzyme. The fourth, with hepatomegaly and no hypoglycaemic symptoms, had a normal glucose-6-phosphatase enzyme but a defect in the hepatic microsomal phosphate/pyrophosphate translocase T2; this is the first report of an adult with type 1c GSD. Adult type 1 GSD should be considered in patients with unresolved hypoglycaemic symptoms and/or unresolved hepatomegaly.

Propranolol in chronic liver disease: a controlled trial of its effect and safety over twelve months.

The effect and safety of propranolol (Inderal-LA) in 95 patients with mild to moderate chronic liver disease was studied in a double-blind placebo controlled trial. Over a 12-month period, three patients in the propranolol group died compared with eight in the control population. Upper gastrointestinal bleeding occurred in the placebo group only. Twenty-five patients were withdrawn, 12 in the propranolol and 13 in the placebo group. No deterioration in clinical condition or liver function tests was observed in the propranolol-treated patients, although serum testosterone levels fell significantly compared with the control patients. This study shows that long-term treatment with propranolol is safe in patients with chronic liver disease but further studies are required to define whether or not patients will benefit. Our observations on the response to placebo suggest that a significant proportion of patients are not likely to tolerate drug treatment for portal hypertension well.

Pulmonary manifestations of Whipple's disease.

Whipple's disease is a rare multisystem disorder which may affect the lungs and pleurae. Four men with Whipple's disease are described. All developed dyspnoea and pleuritic chest pain and three had a chronic cough. Lung function tests in three patients showed reduced lung volumes. Chest radiographic changes included pleural adhesions, small lung volumes and nodular shadowing. In three patients treatment of the Whipple's disease resulted in resulted in resolution of the disabling breathlessness, thus underlining the importance of recognising this feature of the disease. Although intestinal and joint manifestations of Whipple's disease are attributed to intracellular infection, the respiratory manifestations may be due to an inflammatory reaction to locally deposited immune complexes containing bacterial antigens.

Primary biliary cirrhosis, dark adaptometry, electro-oculography, and vitamin A state.

Twenty five patients with primary biliary cirrhosis were studied for vitamin A state. In nine patients found to have low circulating vitamin A concentrations no abnormality was found on electro-oculography or in dark adaptation. A positive correlation was found between retinol binding protein and vitamin A values (r = +0.88; p less than 0.001) and between serum albumin and vitamin A values (r = +0.75; p less than 0.001). A weaker and negative correlation was found between serum bilirubin (r = -0.47; p less than 0.05) and vitamin A values. Patients with primary biliary cirrhosis should not receive regular parenteral or even oral vitamin A supplementation unless dark adaptometry or electrooculography yields an abnormal result.