Cryopreservation of quince (Cydonia oblonga Mill.).

BACKGROUND: Quince (Cydonia oblonga Mill.) has great potential for utilisation in pharmaceutical and food industries. OBJECTIVE: The study was to develop an efficient cryopreservation approach for quince. METHODS: Factors on the survival and regrowth such as cold acclimation, explant type and recovery media composition were assessed. The effectiveness of the resultant protocols for a number of quince cultivars was determined. RESULTS AND CONCLUSION: Quince shoot tips and nodal sections are successfully cryopreserved. Sustained regrowth of quince Angers A was observed after encapsulation-osmoprotection/dehydration, encapsulation-dehydration and PVS2 vitrification. The highest regrowth rate (80%) was obtained from explants excised from cold hardened shoots and cryopreserved using encapsulation-osmoprotection/dehydration and vitrification protocols. The optimised vitrification protocol in combination with shoot cold hardening and a MS recovery medium without activated charcoal and auxin resulted in satisfactory regrowth of shoots from six quince cultivars. The morphology of acclimatised plants derived from cryopreserved shoots was comparable with non-cryopreserved plants.

Cryopreservation of redwood (Sequoia sempervirens) in vitro buds using vitrification-based techniques.

In this study, the efficiency of three vitrification-based cryopreservation techniques, i.e. vitrification, encapsulation-vitrification and droplet-vitrification were compared for cryopreserving Sequoia sempervirens apical and basal buds sampled from in vitro shoot cultures. The effect of cold-hardening of mother-plants and of bud culture medium and sucrose preculture was also investigated. Culture of apical and basal buds sampled from cold-hardened mother-plants on Quoirin and Lepoivre medium with activated charcoal had a positive effect on regrowth. Only droplet-vitrification ensured survival and regrowth after cryopreservation. After cryopreservation, regeneration of apical buds was possible for PVS2 exposure durations between 90 and 180 min but it remained low, with a maximum of 18 percent after 135 min treatment. With basal buds, regeneration after cryopreservation was possible over a larger range of PVS2 treatment durations, between 30 and 180 min. The highest regeneration percentage was slightly higher (22 percent) than that measured with apical buds, and was also achieved after 135 min PVS2 exposure.

Angular-overlap analysis of the iron(II) site in [2Fe-2S] clusters.

The angular-overlap model for Fe(II)S4 centres is used to obtain structural information form the experimental data available for the gav approximately equal to 1.96 and gav approximately equal to 1.91 classes of [2Fe-2S] ferredoxin, showing that it is possible to translate the parameters obtained by Bertrand and Gayda in their non-additive ligand field model (Bertrand, P. and Gayda J.P. (1979 and 1980) Biochim. Biophys. Acta 579, 107-121 and 625, 337-342, respectively) into an additive one. The analysis of the e lambda (lambda = sigma or pi) parameters allowed us to conclude that the Fe(II)S4 chromophores of the two types of [2Fe-2S] metallo-protein are similar to each other, being possible to reproduce nicely the different g tensors introducing only small variations in the angular and bonding parameters.

Changes in low-Km cAMP phosphodiesterase activity in liver Golgi fractions from hyper- and hypoinsulinemic rats.

Acute insulin treatment in rats has recently been shown to cause a rapid increase in liver low-Km cAMP phosphodiesterase (PDE) activity, which selectively affects Golgi fractions. To assess the physiological significance of this observation, the cAMP PDE activity associated with liver Golgi fractions has been measured in genetically obese Zucker rats, which spontaneously develop hyperinsulinemia, in rats receiving a continuous infusion of insulin, and in rats treated with anti-insulin serum. In genetically obese Zucker rats, a significant increase in Golgi-associated cAMP PDE relative to age-matched lean animals occurred after 3 wk, coinciding with the development of hyperinsulinemia. This change was maximal at 5-8 wk and affected the light (Gl) and intermediate (Gi) Golgi fractions (100-110% increase) to a greater extent than the heavy (Gh) fraction (30% increase). After 7 wk, despite the further increase in insulinemia, the increase in Golgi-associated cAMP PDE became progressively less marked, and at 18 wk it was no longer detectable except in Gh, suggesting the development of a hepatic insulin resistance. Infusion of insulin through chronically implanted intracardiac catheters led to a 30-50% increase in Golgi-associated cAMP PDE, which occurred earlier in Gi (3 h) than in Gh (7 h) and persisted for greater than 96 h. Injection of anti-insulin serum led to a 30-50% decrease in Golgi-associated cAMP PDE, which occurred sequentially in Gl (5 min), Gi (15 min), and Gh (30 min) and affected predominantly Gl and Gh. These results suggest that the cAMP PDE associated with Golgi fractions is a physiological effector of plasma insulin in vivo.

Characterization of a liver low Michaelis-Menten constant 3',5'-cyclic adenosine monophosphate phosphodiesterase activity sensitive to thyroid status.

These studies were undertaken to assess the subcellular distribution and some biochemical properties of the hepatic cAMP phosphodiesterase(s) whose activity is modulated by the thyroid status in the rat. Thyroidectomy led to a 2-fold increase in low Michaelis-Menten constant (Km) cAMP phosphodiesterase activity in Golgi-endosomal fractions, but little affected this activity in crude particulate fractions. On analytical sucrose density gradients, an increase in cAMP phosphodiesterase activity in particulate elements which equilibrated at densities 1.17-1.22 was also observed. Acute insulin treatment did not further increase cAMP phosphodiesterase activity in Golgi-endosomal fractions of thyroidectomized rats. Up to 75% of the cAMP phosphodiesterase activity associated with Golgi-endosomal fraction of euthyroid and hypothyroid rats was inhibited by cGMP (IC50, 10 microM and 1 microM, respectively). Activity was also potently inhibited by griseolic acid, cilostamide, and cilostazole (IC50, less than 1 microM) but was much less sensitive to R0-20-1724 (IC50, 1 mM). Treatment of Golgi-endosomal fractions by a hypotonic extract of rat liver lysosomes led to the solubilization of 50% of low Km cAMP phosphodiesterase activity. On sucrose density gradients, the solubilized activity migrated as a slightly asymmetrical peak with a sedimentation coefficient of 6 S in euthyroid rats and 6.9 S in hypothyroid rats. On nondenaturing polyacrylamide gel electrophoresis, the activity migrated as two majors peaks with Rf values of 0.23 and 0.50; only the activity associated with the fast-moving peak was increased by thyroidectomy. On diethylaminoethyl-Sephacel chromatography, four peaks of cAMP phosphodiesterase activity, two of which were cGMP-inhibitable, were resolved. Thyroidectomy increased the activity associated with one of the cGMP-inhibitable peaks (eluted at 0.7-0.9 M sodium acetate) and led to the appearance of a new peak of activity (eluted at 0.4 M), which was not sensitive to cGMP. These results show that the low Km phosphodiesterase activity associated with liver Golgi-endosomal fractions, previously shown to be increased in hyperinsulinemic rats, is also increased in hypothyroid animals. They also suggest that, based on pharmacological and physical criteria, the enzyme species affected by the thyroid status belongs to the cGMP-and cilostamide-inhibited subclass of low Km cAMP phosphodiesterases.

Pyruvate dehydrogenase activity is stimulated by growth hormone (GH) in human mononuclear cells: a new tool to measure GH responsiveness in man.

Human peripheral mononuclear cells (PMC) were used to examine the effects of hGH and insulin on the activity of the pyruvate dehydrogenase (PDH) complex. Incubation of PMC with 10(-7) mol/L hGH or insulin increased basal PDH activity. Hormonal effects were maximal (50-60% above control values) at 15 min. Later on, activation progressively decreased and was no longer detectable at 30 min. Total PDH activity was unaffected by hormonal treatment. PMC were subfractionated into lymphocytes and monocytes to assess the sensitivity of each cell types to the hormones. hGH significantly increased basal PDH activity in lymphocytes and monocytes (38% and 70% above control values, respectively), whereas insulin increased basal PDH activity only in monocytes (151% above control value). PMC from healthy subjects aged 1-45 yr were incubated for 15 min with 10(-7) mol/L hGH or insulin before PDH measurement. An increase of enzyme activity higher than 20% was observed in 26 patients out of 29 with hGH, and in 15 out of 18 with insulin. In conclusion, hGH is able to stimulate PDH activity of human mononuclear cells. This hormonal effect allows rapid evaluation of the cellular responsiveness of hGH in various pathophysiologic situations.

Nonparallel changes of growth hormone (GH) and insulin-like growth factor-I, insulin-like growth factor binding protein-3, and GH-binding protein, after craniospinal irradiation and chemotherapy.

We studied the GH-insulin-like growth factor-I (IGF-I) axis serially over 24-36 months in six patients with medulloblastoma who underwent surgical removal of the tumor followed by craniospinal irradiation therapy for 6 weeks and then chemotherapy for 42 weeks. Eighteen and 24 months after beginning irradiation there was a decline in the peak GH secretory response to acute stimulation with arginine/insulin hypoglycemia. Six months after irradiation and during chemotherapy there was a transient decline in IGF-I, IGF binding protein-3 (IGFBP-3), and GH-BP values (respective mean values of 56.1 +/- 9.0 ng/mL, 1.1 +/- 0.2 microgram/mL, and 7.6 +/- 3.3% of radioactivity as compared to time 0 values: 13%%o/- 15 ng/mL, 2.2 +/- 0.2 micrograms/mL, and 20.0 +/- 4.0%, P < 0.001), although provoked GH secretion was normal at this time. The IGF-I, IGFBP-3, and GH-BP returned to pretreatment ranges by 12-36 months after initiation of the study. There was also a decline in body mass index and serum protein values at 6 months, suggesting suboptimal nutrition during this period. Six months after irradiation in ligand and immunoblot analysis there was a decline in IGFBP-3 and an abnormal electrophoretic mobility of IGFBP-2 that were both normalized at 36 months. In one patient we observed a high level of IGFBP-3 proteolysis at this time. This study demonstrates that before the decrease of GH secretion in patients receiving cranial irradiation there is a transient phase of GH insensitivity that may be characteristic of the acute therapeutic phase including the chemotherapy. This partial insensitivity may explain the early growth retardation observed in these patients.

Longitudinal study of tissue- and subunit-specific obesity-induced regulation of the pyruvate dehydrogenase complex.

The tissue-specific expression of the mitochondrial pyruvate dehydrogenase complex (PDHc) has been studied in an animal model of obesity with hyperinsulinemia, the obese (fa/fa) Zucker rat. Liver and heart were obtained from 4 and 8 week-old obese rats and age-matched lean animals, and in each tissue the following parameters were analyzed: (1) total activity of the mitochondrial PDHc; (2) abundance of the mitochondrial PDHc subunits on Western blots; and (3) abundance of the E1alpha and E1beta subunit mRNAs on Northern blots and semi-quantitative RT-PCR. Regardless of age, obese rats showed an increase in liver total PDHc activity and a coordinate increase in liver E1alpha and E1beta PDHc subunit abundance. At 4 weeks, obese rats also showed an increase in liver PDH E1alpha mRNA level, but regardless of age E1beta mRNA level was unchanged. In contrast, neither total PDHc activity nor the concentration of its protein subunits were increased in heart of obese rats. Thus, obese Zucker rats display a liver-specific early increase in PDHc which results from a selective up-regulation of the E1alpha gene expression.

A family of polynuclear cobalt and nickel complexes stabilised by 2-pyridonate and carboxylate ligands

The synthesis and structural characterisation of a series of cobalt and nickel cages are reported. Eight of these structures contain a [M10(mu3-OH)6(eta2, mu3-xhp),(eta2, mu2-O2CR)6]2+ core (where M = Co or Ni; xhp = 6-chloro- or 6-methyl-2-pyridonate: R = Me, Ph, CHMe2, CH2Cl, CHPh2 or CMe3), where the ten metal atoms describe a centred-tricapped-trigonal prism (ttp). The cage contains six hydroxide ligands around the central metal, and the exterior is coated with pyridonate and carboxylate ligands. For four of the cages additional metal centres are found attached to the upper and/or lower triangular faces of the trigonal prism, generating dodeca- and undecanuclear cages. Three further cages are reported that contain a metal core based on an incomplete centred-tetraicosahedron. These cages involve trimethylacetate as a ligand in company with either 6-methyl-2-pyridonate or 6-chloro-2-pyridonate. Comparison of these latter structures with the trigonal prisms reveal that they can be described as a pentacapped-trigonal prism missing one edge. Magnetic studies of three of the nickel cages with trigonal prismatic cores show spin ground states of S = 8, 4 and 2 for Ni12, Ni11 and Ni10 cages, respectively.

Evidence for a role of protein kinase C in the activation of the pyruvate dehydrogenase complex by insulin in Zajdela hepatoma cells.

The signal transduction pathway involved in the activation of pyruvate dehydrogenase (PDH) by insulin is still unknown. In this study, we have examined the possible involvement of protein kinase C (PKC) in the process. In addressing this question, we examined (1) the insulin-like effects of the PKC activator 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) on the PDH complex, (2) the effects of various PKC inhibitors on the PDH activation by insulin, and (3) the response of PKC-depleted cells to insulin. We used as an experimental model Zajdela hepatoma cultured (ZHC) cells, which have been demonstrated to be responsive to physiological doses of insulin. Half-maximal and maximal stimulations of the PDH complex by insulin were observed at 0.05 and 5 nmol/L, respectively. Stimulation of PDH activity by insulin (5 nmol/L) occurred within 5 minutes of incubation and was maximal (+70%) at 7.5 minutes. In the presence of PMA (162 nmol/L), enzyme activity increased within 30 seconds, was maximal (+90%) at 5 minutes, and was no longer detectable after 10 minutes. Total PDH activity was unchanged by insulin or PMA treatment. The effects of PMA and insulin on basal PDH activity were not additive. Moreover, various inhibitors of PKC--staurosporine, sphingosine, acridine orange--completely blocked the stimulation of PDH activity induced by insulin or PMA. A 17-hour treatment of ZHC cells with 500 nmol/L PMA efficiently downregulated PKC, as attested by the marked decrease in the enzyme activity and the loss of phorbol 12,13-dibutyrate binding to intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of growth hormone on pyruvate dehydrogenase activity in intact rat liver and in isolated hepatocytes: comparison with insulin.

The effects of growth hormone and insulin on the activity of pyruvate dehydrogenase were examined in the rat, both in vivo and in isolated hepatocytes. Liver mitochondria isolated from rats killed from five to 45 minutes after injection of 50 micrograms/100 g human growth hormone (hGH) or 25 micrograms/100 g insulin displayed a significant increase in the activity of basal pyruvate dehydrogenase (38% and 48% above control at ten minutes, respectively). These changes probably result from the conversion of the phosphorylated form to the nonphosphorylated form of pyruvate dehydrogenase since total enzyme activity was unaffected. Treatment of isolated hepatocytes by hGH or insulin also led to an increase in pyruvate dehydrogenase activity which was maximal (25% above control value) at 15 minutes. Later, activation progressively decreased and was no longer detectable at 60 minutes. The concentrations of hGH or insulin required for maximal activation were 100 nmol/L and 20 nmol/L, respectively, and the concentration required for half-maximal stimulation was 2 nmol/L for both hormones. The effects of 100 nmol/L hGH and 100 nmol/L insulin on pyruvate dehydrogenase activity were not additive. Basal pyruvate dehydrogenase activity in hepatocytes exhibited linear kinetics; hGH or insulin increased the Vmax of the enzyme without changing its Km and did not affect the Vmax of the total enzyme activity. It is concluded that growth hormone is as potent and as efficient as insulin in its ability to stimulate the activity of liver pyruvate dehydrogenase, and thus may be a physiological activator of this enzyme.

Leigh syndrome: pyruvate dehydrogenase defect. A case with peripheral neuropathy.

Pyruvate dehydrogenase deficiency is one of the most common causes of encephalopathy associated with lactic acidosis and is known to account for congenital lactic acidosis, recurrent ataxia, and infantile Leigh syndrome. Hitherto, however, peripheral neuropathy has not been regarded as a presenting symptom of pyruvate dehydrogenase deficiency. Here, we report on a boy who presented peripheral neuropathy with severe limb hypotonia, absent deep-tendon reflexes, and reduced motor nerve conduction velocities at 8 months of age. Persistent hyperpyruvicemia with normal lactate/pyruvate molar ratios in plasma were highly suggestive of a pyruvate dehydrogenase deficiency, and the determination of pyruvate dehydrogenase activity in circulating lymphocytes led to the diagnosis of pyruvate decarboxylase (PDH-E1) deficiency in the proband. Based on this observation, we suggest that pyruvate dehydrogenase deficiency should be considered in the diagnosis of peripheral neuropathy in infancy, especially when associated with persistent hyperpyruvicemia, normal lactate/pyruvate molar ratios in plasma, and recurrent episodes of drowsiness and hypotonia of unknown origin.

Reproducibility of a dermoscopic method (7FFM) for the diagnosis of malignant melanoma.

Dermoscopy improves sensitivity in the diagnosis of melanoma. We have developed a new diagnostic dermoscopic method which evaluates only seven dermoscopic features or criteria. We called the method Seven features for melanoma (7FFM). To present and to evaluate the reproducibility of our dermoscopic diagnostic method (7FFM) we held eight dermoscopic courses from 10/4/96 to 04/03/98 in various Italian cities. In fact the reliability of a diagnostic test or method mainly depends on the level of agreement in the interpretation of results among different observers. Only methods with good agreement can be used in clinical practice for the diagnosis of melanoma. Many dermatologists (207) attended one of the eight dermoscopic courses: each course was one-day in length and at the end of the course the participants evaluated a set of 25 dermoscopic slides using our dermoscopic method. Percentages of concordance and K values for a kappa statistical analysis to evaluate inter-rater reliability have been calculated. The method showed a mean percentage of concordance of 85.7%, median 88%, a mean K value of 0.699, median 0.684. These data point to a good agreement level. Our method shows good reproducibility after a short training program.

The seven features for melanoma: a new dermoscopic algorithm for the diagnosis of malignant melanoma.

The clinical diagnosis of melanoma has a mean sensitivity of 67%, dermoscopy or dermatoscopy is a non invasive technique which improves this sensitivity. Our purpose was to create a simple dermoscopic method for the diagnosis of melanoma useful in daily office practice. For this reason a training set of 218 cutaneous pigmented lesions was used and scored for 16 dermoscopic features: for each feature sensitivity, specificity and statistical significance were evaluated. The results were used to create a simple dermoscopic diagnostic method of only seven dermoscopic features (7FFM). The method was used to evaluate a test set of 713 pigmented skin lesions consecutively observed. The diagnostic dermoscopic method developed gave a sensitivity of 94.6%, a specificity of 85.5% and an efficiency of 87.6%. Our method improves the sensitivity in the diagnosis of melanoma and can be used for the screening of pigmented skin lesions.

Reproducibility of the clinical criteria (ABCDE rule) and dermatoscopic features (7FFM) for the diagnosis of malignant melanoma.

Dermatoscopy improves the sensitivity and the specificity in the diagnosis of melanoma. Although the reproducibility of dermatoscopic features has been the subject of research, no study up to now has compared the reproducibility of dermatoscopic features to the reproducibility of the clinical criteria of the ABCDE rule. For this reason we decided to examine the reproducibility of the clinical ABCDE rule and of our diagnostic dermatoscopic method 7FFM, as well as of the individual criteria of both. A total of 73 dermatologists attended three dermatoscopic courses and examined a set of clinical and dermatoscopic slides of 50 pigmented skin tumors. Agreement % and K value for a kappa statistical analysis have been calculated to evaluate inter-rater reliability. The clinical and the dermatoscopic methods showed similar values of concordance: clinical score 2 mean agreement = 68%, mean K = 0.44; clinical score 3 mean agreement = 73%, mean K = 0.61; 7FFM mean agreement = 83%, mean K = 0.64. The clinical criteria A, B, and C and the dermatoscopic features of our method presented similar values of concordance as well: clinical criteria mean K range 0.35-0.25, dermatoscopic features mean K range 0.62-0.25. The dermatoscopic features of our method 7FFM show a good reproducibility after a short training program, similar to the reproducibility of the clinical criteria of the ABCDE rule for the diagnosis of melanoma.

The dermoscopic versus the clinical diagnosis of melanoma.

Dermoscopy is an useful technique for the diagnosis of melanoma. All the diagnostic dermoscopic methods developed until now have been tested on dermoscopic slides and the real improvement given by this technique to the clinical diagnosis based upon ABCDE criteria is still unknown. For this reason, we decided to undertake a prospective study to compare the clinical diagnosis made with ABCDE criteria, to the dermoscopic diagnosis, made with the method 7FFM, developed by us. A total of 401 lesions were evaluated clinically and dermoscopically. On the basis of the number of the clinical criteria considered sufficient to diagnose a melanoma, various clinical scores, ranging from 1 to 5, can be obtained; data about the sensitivity of the clinical diagnosis of melanoma suggest that the most often used score is score 3. Our method 7FFM presents a sensitivity and a specificity better than those obtained with score 3: 80% versus 66.6% and 89.1% versus 79.3%. Besides this, we have evaluated if the sensitivity of our method 7FFM in the diagnosis of melanoma can be improved with the adjunct of the clinical criteria or of the clinical scores. The best values of sensitivity 93.3% and predictive value negative 97.3% have been obtained with score 2 plus 7FFM. Our results confirm that our method can be used in the screening of pigmented skin lesions in daily office practice and that an improvement in sensitivity without an excessive sacrifice of specificity can be obtained with the adjunct of the clinical score 2.

Hemostatic efficacy and safety of TachoComb in surgery. Ready to use and rapid hemostatic agent.

TachoComb is a new, ready-to-use hemostatic agent consisting of a collagen sheet coated on one side with human fibrinogen, bovine thrombin, and bovine aprotinin. The product was used in 125 surgical operations (vascular, hepatic, urological and ENT) in which secondary hemostasis was required. It was placed over the cut surface or over the edges of the wound. The investigating surgeons expressed their opinion on the intra- and postoperative hemostatic efficacy, and routine laboratory tests were done postoperatively. TachoComb had good hemostatic efficacy in 67.2% of cases, and very good in 22.4%. No noteworthy systemic changes were observed. As an adjuvant to obtain complete hemostasis in surgery, TachoComb is effective, practical and quick to use, and is very well tolerated.

Development of a shoot-tip vitrification protocol and comparison with encapsulation-based procedures for plum (Prunus domestica L.) cryopreservation.

Cryopreservation of plum (Prunus domestica L.), cv Regina Claudia, was obtained by a vitrification/one-step cooling procedure of shoot tips from cold-hardened in vitro-grown plants. Best survival (57%) was obtained when the shoot tips were precultured at 4 degree C for 2 days on 0.09 M sucrose-containing Quoirin and Le Poivre medium, loaded for 30 min with a cryoprotectant (2 M glycerol and 0.4 M sucrose), incubated with the PVS2 solution at 0 C for 90 min, and directly plunged into liquid nitrogen. After re-warming in a waterbath at 40 degree C, the shoot tips were washed in a 1.2 M-sucrose MS solution for 20 min and finally plated on a regrowth medium. In comparison with the one-step cooling procedure, both the slow cooling (-0.5 degree C/min up to -45 degree C), and the two-step cooling (-160 degree C for 25 min, then -196 degree C) gave lower percentages of shoot-tip survival. Among the other cryogenic procedures tested, the performance of the encapsulation-vitrification method was similar to the vitrification protocol in terms of shoot-tip regrowth (47.5%), while encapsulation-dehydration was unsatisfactory.