Agrobacterium-mediated transformation of Solanum phureja.

A population of transgenic plants was produced by the transformation of internodal explants of Solanum phureja, DB337/37 (the cultivar Mayan Gold) using an Agrobacterium tumefaciens LBA4404-based vector containing a phytoene synthase gene (crtB). The regeneration strategy utilised a two-step protocol, with a 12-day callus induction stage mediated by 1.07 microM alpha-napthaleneacetic acid (NAA), 7.10 microM zeatin riboside and 0.06 microM gibberellic acid (GA3), followed by a prolonged (up to 90 day) shoot induction stage on medium containing 0.11 microM NAA, 7.10 microM zeatin riboside and 0.06 microM GA3 supplemented with kanamycin at 50 mg l(-1) as the selection agent. Southern analysis of the transgenic population revealed that the transgene copy number varied between one and five in the lines tested. Northern blot analysis showed significant expression of the introduced crtB gene in some lines during tuber development. Cytological analysis of the material showed a high incidence of chromosome doubling in the transgenic population with over 80% of all lines tested having doubled their chromosome complement during the transformation process.

Carotenogenesis during tuber development and storage in potato.

Germplasm of Solanum tuberosum and Solanum phureja exhibit a wide (over 20-fold) variation in tuber carotenoid content. The levels of carotenoids during tuber development and storage were compared in a high carotenoid-accumulating S. phureja accession (DB375\1) with two S. tuberosum cultivars (Pentland Javelin and Desiree) that accumulate lower levels of tuber carotenoid. On a dry weight basis, total carotenoid levels were at a maximum early in tuber development. However, in the S. phureja accession, carotenoid levels remained at a high level throughout tuber development, whereas in the S. tuberosum accessions, carotenoid content decreased as dry weight increased. The carotenoid profiles of tissues during tuber development were analysed in greater detail by reverse phase HPLC. In S. phureja tubers at maturity the major carotenoids were zeaxanthin, antheraxanthin, and violaxanthin. Following 9 months storage at 4 degrees C the levels of zeaxanthin and antheraxanthin decreased, whereas the level of lutein increased; overall, however, there was only a small decrease in total carotenoid content. In order to explore the reasons for the wide variation in tuber carotenoid content, the expression patterns of the major genes encoding the enzymes of the carotenoid biosynthetic pathway were compared. Significant differences in the profiles were detected, suggesting that transcriptional control or mRNA stability gives rise to the large differences in tuber carotenoid content. In particular, there was an inverse trend between the level of zeaxanthin epoxidase transcript level and tuber carotenoid content in a range of potato germplasm, giving rise to an hypothesis for the regulation of carotenogenesis in potato tubers.

Molecular analysis of myeloperoxidase deficiency shows heterogeneous patterns of the complete deficiency state manifested at the genomic, mRNA, and protein levels.

Myeloperoxidase (MPO) is a glycosylated hemoprotein contained in the azurophil granules of human polymorphonuclear leukocytes (PMNs). MPO is thought to play a role in the oxidative antimicrobial activity of neutrophils by catalyzing the formation of hypochlorous acid, a potent microbicide, from hydrogen peroxide and chloride anions. Seven unrelated individuals with complete MPO deficiency, a relatively common heritable defect of neutrophils, were identified during routine blood tests. Molecular analyses were conducted to determine the level of the abnormality in these individuals. Western blot analysis showed that 6 of the 7 donors were devoid of immunoreactive MPO protein, while neutrophils from one individual contained only the 55-Kd subunit. Northern analysis of bone marrow RNA from one MPO-deficient donor showed the presence of the normal-sized 3.3-kb transcript indicating that the defect in MPO biosynthesis in this case was posttranscriptional. Southern analysis of four MPO-deficient donors showed a normal Bgl II digestion pattern, whereas an abnormal restriction pattern was observed in a fifth individual. Although the Bgl II pattern was similar to that observed in an unrelated subject described by Nauseef (Blood 73:290, 1989), our study strongly suggests that the point mutation does not reflect a polymorphism. Taken together, these analyses show the existence of diverse abnormalities associated with MPO-deficiency that may be detected at the level of the MPO polypeptide, mRNA, and gene.

Enterohepatic regulation and metabolism of 3,5,3'-triiodothyronine in hypothyroid rats.

Several steady state indices of thyroid hormone distribution, metabolism, excretion, and absorption were measured in intact hypothyroid and euthyroid rats, to explore the role of intestines and enterohepatic pathways in the dynamic regulation of whole-body thyroid hormone in these two states. Ten rats were studied, 5 normal control (N) and 5 rendered hypothyroid (3.48 vs. 19.8 ng/ml TSH) by surgical thyroidectomy 3.5 weeks earlier (HYPO). High specific activity 125I-labeled T3 (T3*) was infused at the same constant rate for 7 days from osmotic minipumps implanted sc. Daily urine and feces, and seventh-day cardiac and portal venous blood, bile, and whole intestinal contents were assessed. Bowel and feces were homogenized, extracted, and chromatographed, along with serum, bile, and urine samples. Bile, bowel, and fecal extract samples were also hydrolyzed with aryl-sulfatase and/or beta-glucuronidase and chromatographed to identify conjugates and determine total T3* in all fluid and tissue samples. In the N group, the bowel contained 21.2 +/- 1.22 (SD) times more T3* (mass) than plasma (199 ng vs. 9.39 ng), this ratio falling to 9.03 +/- 1.78 in the HYPO group (30.4 ng vs. 3.37 ng), a shift to relatively more T3* in blood. Urinary T3* was zero in both groups. But fecal excretion was 34 +/- 4.43% of total T3* infused (production) in N and only 20.3 +/- 3.05% in HYPO rats, closely paralleling reduced fecal bulk flow, and thus providing more time for T3* absorption. Endogenous T3 and T4 concentrations measured in portal plasma were 15-31% greater in normals and 69-95% greater in HYPO rats than in corresponding systemic plasma samples, a direct indication of absorption of endogenous T3 and T4 in both groups, with greater absorption in the HYPO group. About 66% total T3* was metabolically degraded in N rats, rising to approximately 80% in HYPO rats. Plasma clearance rates of T3 fell more than 50% in HYPO rats, and total T3 production fell to about 20% of normal. It appears that HYPO rats compensate for low T3 by fecally excreting a much smaller fraction of total T3 production, absorbing more T3 and T4, and leaving a larger fraction for T3 action and degradative metabolism.

Purification, primary structures, and antibacterial activities of beta-defensins, a new family of antimicrobial peptides from bovine neutrophils.

A new family of cysteine-rich antimicrobial peptides from bovine neutrophils was isolated and characterized. Thirteen structurally homologous peptides were purified to homogeneity from a granule-rich cytoplasmic fraction of purified blood neutrophils. The complete sequences of the peptides were determined by a combination of enzymatic digestion, Edman degradation, and additional biochemical characterization of the carboxyl termini. The peptides are characterized by a highly cationic 38-42-residue chain which includes 6 invariantly spaced cysteines which form three disulfides. They share a highly conserved consensus sequence which is also found in a recently described epithelial antimicrobial peptide from bovine trachea. The in vitro antibacterial activities of the 13 neutrophil peptides, determined in assays using Staphylococcus aureus and Escherichia coli as test organisms, demonstrated that each peptide possessed antimicrobial activity, and that several were as active as the most potent neutrophil defensin, rabbit NP-1. Though the structural and functional attributes of the bovine neutrophil peptides are similar to those of defensins, the two peptide families are distinguished by their unique consensus sequences and additionally by differing tridisulfide motifs. We therefore propose that this new defensin-like antimicrobial peptide family be named beta-defensins.

Indolicidin, a novel bactericidal tridecapeptide amide from neutrophils.

A potent and structurally novel antimicrobial peptide was purified from the cytoplasmic granules of bovine neutrophils. Suspensions of Staphylococcus aureus and Escherichia coli were virtually sterilized by the peptide at a concentration of 10 micrograms/ml. The peptide was found to be comprised of 13 amino acids, 5 of which were tryptophan residues, and the carboxyl-terminal arginine was carboxamidated. The primary structure of the peptide, which we have named indolicidin, is H-Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-NH2. The mole percent of tryptophan in indolicidin is the highest observed among known protein sequences. The multiple tryptophan residues presumably play an important role in the function of this unique antibiotic peptide.

Insulin binding to the blood-brain barrier in the streptozotocin diabetic rat.

125I-Insulin binding to isolated brain microvessels from control, streptozotocin diabetic, and insulin-treated diabetic rats was measured. The binding was highest in the control (21.1 +/- 1.8%/mg capillary protein) and lowest in the diabetic (14.8 +/- 1.9%, p less than 0.01) animals. Administration of 2 U of protamine zinc insulin per day increased the maximum binding in the diabetic rats to 17.2 +/- 2.1%. Scatchard analyses of the binding showed that the major difference between the diabetic and the control animals was a decrease in the number of both high- and low-affinity sites in the diabetic animals. To test whether the failure of up-regulation in the hypoinsulinemic diabetic animal was related to an inherent defect in the endothelial cell or resulted from the diabetic milieu, cultured brain endothelial cells were tested for their capacity to up- and down-regulate their insulin receptors in vitro. In response to 100 ng/ml insulin for 12 h, these cells down-regulated their insulin receptors. When the insulin was removed, the insulin receptors returned to control levels. These studies showed that in vitro brain capillary endothelial cells have the capacity to increase their insulin receptors in response to a low-insulin environment, whereas in vivo the microvessels decrease their insulin receptors in response to diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding and internalization of insulin and insulin-like growth factors by isolated brain microvessels.

Isolated brain capillaries were used as a model system to test for binding and internalization of insulin and insulin-like growth factors (IGF) I and II. At 37 degrees C, the maximum specific binding of the 125I-labeled peptides was 48.0 +/- 0.8%/mg capillary protein for IGF I, 40.6 +/- 1.4% for IGF II, and 15.1 +/- 0.6% for insulin. The concentration of unlabeled peptide needed to cause a 50% decrease in the maximum binding (ID50) was 22 ng/ml (2.9 nM), 25 ng/ml (3.3 nM), and 7 ng/ml (1.2 nM) for IGF I, IGF II, and insulin, respectively. Unlabeled insulin competed poorly for the IGF I receptor, requiring 5000 ng/ml (667 nM) to cause a 50% reduction in binding, and did not compete at all for the IGF II receptor at concentrations up to 10(5) ng/ml (17.8 microM). The IGF I receptor was further characterized by reduced polyacrylamide gel electrophoresis of the disuccinimidyl suberate cross-linked 125I-labeled IGF I receptor. The gel showed a distinct band at 133,000 Mr that was abolished by 0.6 microgram/ml (80 nM) unlabeled IGF I but not by 10.0 micrograms/ml (1780 nM) unlabeled insulin. Peptide internalization was monitored by the acidwash technique. Only 22% of the bound IGF I was internalized, but 50% of the insulin and 43% of the IGF II were acid resistant. Capillaries prelabeled with internalized 125I-insulin could then export radioactivity into fresh, insulin-free media in a time- and temperature-dependent manner. However, high-performance liquid chromatography (HPLC) and trichloroacetic acid (TCA) analysis of the released material showed that it consisted mostly of degraded peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of glyburide on in vivo recycling of the hepatic insulin receptor.

Sulfonylureas affect insulin action at both receptor and post-receptor sites, but their exact mechanism is poorly understood. In these studies, a novel technique was used to examine the influence of glyburide on in vivo cycling of the hepatic insulin receptor. Rats were gavage-fed with 5 mg/kg per day of glyburide solubilized in 60 percent polyethylene glycol and 40 percent phosphate buffer. Control rats were fed polyethylene glycol and buffer alone. After seven days, each rat was anesthetized, the abdomen was surgically exposed, and the animal was given a saturating bolus of 30 micrograms of unlabeled insulin via the portal vein. At seven specified times from 10 seconds to 45 minutes later, a second portal vein injection of a mixture of 1.5 microCi (0.015 micrograms) 125I-labeled insulin and 15 microCi 3HOH (a highly diffusible internal reference marker) was administered; 18 seconds later (time for one circulation), the right lobe of the liver was removed, and 125I and 3H values were counted. The liver uptake index and the turnover half-time were then calculated. Glyburide caused a doubling of the turnover half-time for the receptor. This suggests that sulfonylureas potentiate the action of insulin either by increasing the dwell time of insulin on its receptor or by affecting an intracellular event that delays the recycling of the insulin receptor back to the cell surface plasma membrane. The technique is potentially useful as an in vivo screening assay for the effects of other drugs and hormones on the liver.

Enhanced insulin binding to blood-brain barrier in vivo and to brain microvessels in vitro in newborn rabbits.

Insulin is a known growth factor in nonneural tissue, and recent studies have shown that there are insulin receptors throughout the adult and fetal central nervous system. Since insulin has only limited access to the adult brain, this study was undertaken to determine if insulin has increased availability to the newborn brain where it may act as a neonatal brain growth promoter. In vivo brain uptake of 125I-insulin after a single-pass carotid injection was measured in newborn, 3-wk-old and 11-wk-old (adult) rabbits. The brain uptake index (BUI) relative to a 3HOH reference was 22.0 +/- 1.1% (mean +/- SEM) for newborn, 12.8 +/- 0.6% for 3-wk-old, and 6.5 +/- 0.1% for adults. Specific 125I-insulin binding to isolated cerebral microvessels was similarly increased in the newborn (60.6 +/- 3.3%/mg protein) compared with the 3-wk-old (23.8 +/- 1.7) and adult animals (13.6 +/- 1.9). Scatchard analysis revealed that the difference was due to an increase in receptor number with only minimal changes in the affinity. The increased availability of circulating insulin to the newborn brain was further corroborated by elevated CSF/serum and brain/serum insulin ratios in the newborn versus adult. These results suggest that insulin has increased access to the newborn brain where it may function as a growth factor.