The carbohydrate constituents of the cell wall of suspension cultures of Rosa glauca

Sequential extractions of 14-day-old Rosa glauca cell walls cultured in vitro showed that two different types of acidic polysaccharide were present. One was extracted with EDTA or ammonium oxalate solutions, and the other remained in close association with cellulose even after 4.3 N NaOH extractions or 2 N H₂SO₄ hydrolysis. The cell wall has a low content in structural protein. The behaviour of each constituent sugar was followed during the course of the various extraction steps, and a complete quantitative account of the protein, uronic acid and neutral sugar components is given at each stage.     

Characterization of structurally similar neutral and acidic tetrasaccharides obtained from the enzymic hydrolyzate of a 4-O-methyl-D-glucurono-D-xylan

Two similar tetrasaccharides, one neutral and one acidic, were isolated from the products released by the attack of a xylanase on the in situ reduced 4-O-methyl-D-glucurono-D-xylan from aspen (Populus tremuloides). Paper chromatography, gel filtration behavior, methylation followed by reduction, and mass spectrometry showed that the oligosaccharides were O-(4-O-methyl-α-D-glucopyranosyluronic acid)-(1→2)-D-xylotriose and-O-(4-O-methyl-α-D-glucopyranosyluronic acid)-(1→2)-D-xylotriose. Independent of the acidic or neutral substituent on the present xylan chain, the enzymic cleavage led preferentially to oligosaccharides substituted at the nonreducing end. The existence, in wood, of a few uronic acid substituents of the D-xylan in the esterified form was confirmed, and their linkage to lignin postulated.     

Hemicelluloses of young internodes of Arundo donax

An arabino 4-O-methyl glucurono xylan has been isolated as the dominant form of the hemicellulosic material of the youngest internodes of Arundo donax. The polysaccharide contained d-glucose, d-xylose and l-arabinose in the molar ratios of 0·1:9·2:0·66, respectively, with a uronic acid content of 4·5%. Methylation and periodate-oxidation showed that the xylan has a β-1 → 4-linked xylose backbone and a degree of polymerization ca. 63. The xylan is very similar to that found in the mature tissues.     

Agrobacterium tumefaciens-mediated transformation of leek (Allium porrum) and garlic (Allium sativum)

Transgenic leek (Allium porrum) and garlic (Allium sativum) plants have been recovered by the selective culturing of immature leek and garlic embryos via Agrobacterium-mediated transformation using a method similar to that described by Eady et al. (Plant Cell Rep 19:376–381, 2000) for onion transformation. This method involved the use of a binary vector containing the m-gfp-ER reporter gene and nptII selectable marker, and followed the protocol developed previously for the transformation of onions with only minor modifications pertaining to the post-transformation selection procedure which was simplified to have just a single selection regime. Transgenic cultures were selected for their ability to express the m-gfp-ER reporter gene and grown in the presence of geneticin (20 mg/l). The presence of transgenes in the genome of the plants was confirmed using TAIL-PCR and Southern analysis. This is the first report of leek and true seed garlic transformation. It now makes possible the integration of useful agronomic and quality traits into these crops.     

New insights into the retinal circulation: inflammatory lipid mediators in ischemic retinopathy

Ischemic proliferative retinopathy develops in various retinal disorders, including retinal vein occlusion, diabetic retinopathy and retinopathy of prematurity. Ischemic retinopathy remains a common cause of visual impairment and blindness in the industrialized world due to relatively ineffective treatment. Oxygen-induced retinopathy (OIR) is an established model of retinopathy of prematurity associated with vascular cell injury culminating in microvascular degeneration, which precedes an abnormal neovascularization. The retina is a tissue particularly rich in polyunsaturated fatty acids and the ischemic retina becomes highly sensitive to lipid peroxidation initiated by oxygenated free radicals. Consequently, the retina constitutes an excellent model for testing the functional consequences of membrane lipid peroxidation. Retinal tissue responds to physiological and pathophysiological stimuli by the activation of phospholipases and the consequent release from membrane phospholipids of biologically active metabolites. Activation of phospholipase A(2) is the first step in the synthesis of two important classes of lipid second messengers, the eicosanoids and a membrane-derived phospholipid mediator platelet-activating factor (PAF). These lipid mediators accumulate in the retina in response to injury and a physiologic role of these metabolites in retinal vasculature remains for the most part to be determined; albeit proposed roles have been suggested for some. The eicosanoids, in particular the prostanoids, thromboxane (TXA2) and PAF are abundantly generated following an oxidant stress and contribute to neurovascular injury. TXA2 and PAF play an important role in the retinal microvacular degeneration of OIR by directly inducing endothelial cell death and potentially could contribute to the pathogenesis of ischemic retinopathies. Despite these advances there are still a number of important questions that remain to be answered before we can confidently target pathological signals. This review focuses on mechanisms that precede the development of neovascularization, most notably regarding the role of lipid mediators that partake in microvascular degeneration.     

Urotensin II-induced hypotensive responses in Wistar-Kyoto (Wky) and spontaneously hypertensive (Shr) rats

Human urotensin II (hU-II) is a potent vasoactive peptide which modulates some of the functions of the cardiovascular and other systems. The in vivo mechanism of action by which hU-II may influence blood pressure in developmental and pathological conditions, is poorly understood. Herein, the blood pressure effects of hU-II (0.1-10 nmol/kg) injected intravenously (i.v.) were studied on ketamine/xylazine anesthetized male WKY and SHR rats aged 4 and 8 weeks. hU-II elicited dose-dependent decreases in mean arterial pressure in both strains of animals. The hypotensive responses to hU-II were, however, significantly higher in SHR rats, independently of age. Four-week-old SHR rats (which are normotensive) were, however, less responsive than their hypertensive 8-week-old counterparts. A series of pharmacological inhibitors were used to identify putative endogenous (endothelial) factors that might account for the hU-II-mediated hypotension in 8-week-old SHR. These include the non-selective nitric oxide synthase inhibitor L-NAME (5 micromol/kg), the non-selective cyclooxygenase inhibitor meclofenamate (16 micromol/kg), the voltage-sensitive and ATP-sensitive K+-channel inhibitors, 4-aminopyridine (5 micromol/kg) and glybenclamide (10 micromol/kg), the cytochrome P450 CYP2C9 inhibitor sulfaphenazole (15 micromol/kg), the cytoskeletal fixation agent phalloidin (15 micromol/kg), the endothelin ETB receptor antagonist BQ-788(35 micromol/kg), the bradykinin B2 receptor antagonist HOE 140 (0.5 micromol/kg), the angiotensin AT2 antagonist PD 123319(10 micromol/kg) and the UT receptor antagonist urantide (10 micromol/kg). These agents were administered i.v. either at 2.5, 10 or 40 min prior hU-II injection (10 nmol/kg). Among these inhibitors, sulfaphenazole and phalloidin were able to reduce hU-II-induced hypotension. This suggests that the vasodepressor effect of hU-II is mediated by UT receptors and relies in part on the release of epoxide related products; increased microvascular permeability may also contribute to the blood pressure lowering effect of hU-II. Since urantide blocks the constrictor effects of hU-II on isolated aorta, but is inactive against the hypotensive action of hU-II in vivo, the results presented in this paper provide, for the first time, evidence for the existence of two different functional sites for hU-II.     

Localization of type 8 17beta-hydroxysteroid dehydrogenase mRNA in mouse tissues as studied by in situ hybridization.

The enzyme type 8 17beta-hydroxysteroid dehydrogenase (17beta-HSD) selectively catalyzes the conversion of estradiol (E2) to estrone (E1). To obtain detailed information on the sites of action of type 8 17beta-HSD, we have studied the cellular localization of type 8 17beta-HSD mRNA in mouse tissues using in situ hybridization. In the ovary, hybridization signal was detected in granulosa cells of growing follicles and luteal cells. In the uterus, type 8 17beta-HSD mRNA was found in the epithelial (luminal and glandular) and stromal cells. In the female mammary gland, the enzyme mRNA was seen in ductal epithelial cells and stromal cells. In the testis, hybridization signal was observed in the seminiferous tubule. In the prostate, type 8 17beta-HSD was detected in the epithelial cells of the acini and stromal cells. In the clitoral and preputial glands, labeling was detected in the epithelial cells of acini and small ducts. The three lobes of the pituitary gland were labeled. In the adrenal gland, hybridization signal was observed in the three zones of the cortex, the medulla being unlabeled. In the kidney, the enzyme mRNA was found to be expressed in the epithelial cells of proximal convoluted tubules. In the liver, all the hepatocytes exhibited a positive signal. In the lung, type 8 17beta-HSD mRNA was detected in bronchial epithelial cells and walls of pulmonary arteries. The present data suggest that type 8 17beta-HSD can exert its action to downregulate E2 levels in a large variety of tissues.     

The Human Gonadotropin Releasing Hormone Type I Receptor Is a Functional Intracellular GPCR Expressed on the Nuclear Membrane

The mammalian type I gonadotropin releasing hormone receptor (GnRH-R) is a structurally unique G protein-coupled receptor (GPCR) that lacks cytoplasmic tail sequences and displays inefficient plasma membrane expression (PME). Compared to its murine counterparts, the primate type I receptor is inefficiently folded and retained in the endoplasmic reticulum (ER) leading to a further reduction in PME. The decrease in PME and concomitant increase in intracellular localization of the mammalian GnRH-RI led us to characterize the spatial distribution of the human and mouse GnRH receptors in two human cell lines, HEK 293 and HTR-8/SVneo. In both human cell lines we found the receptors were expressed in the cytoplasm and were associated with the ER and nuclear membrane. A molecular analysis of the receptor protein sequence led us to identify a putative monopartite nuclear localization sequence (NLS) in the first intracellular loop of GnRH-RI. Surprisingly, however, neither the deletion of the NLS nor the addition of the Xenopus GnRH-R cytoplasmic tail sequences to the human receptor altered its spatial distribution. Finally, we demonstrate that GnRH treatment of nuclei isolated from HEK 293 cells expressing exogenous GnRH-RI triggers a significant increase in the acetylation and phosphorylation of histone H3, thereby revealing that the nuclear-localized receptor is functional. Based on our findings, we conclude that the mammalian GnRH-RI is an intracellular GPCR that is expressed on the nuclear membrane. This major and novel discovery causes us to reassess the signaling potential of this physiologically and clinically important receptor.     

Early steps in neural development

We studied early neurulation events in vitro by transplanting quail Hensen's node, central prenodal regions (before the nodus as such develops), or upper layer parts of it on the not yet definitively committed upper layer of chicken anti-sickle regions (of unincubated blastoderms), eventually associated with central blastoderm fragments. We could demonstrate by this quail-chicken chimera technique that after the appearance of a pronounced thickening of the chicken upper layer by the early inductive effect of neighboring endophyll, a floor plate forms by insertion of Hensen's node-derived quail cells into the median part of the groove. This favors, at an early stage, the floor plate "allocation" model that postulates a common origin for notochord and median floor plate cells from the vertebrate's secondary major organizer (Hensen's node in this case). A comparison is made with results obtained after transplantation of similar Hensen's nodes in isolated chicken endophyll walls or with previously obtained results after the use of the grafting procedure in the endophyll walls of whole chicken blastoderms.     

G-protein-coupled receptors signalling at the cell nucleus: an emerging paradigm

G-protein-coupled receptors (GPCRs) comprise a wide family of monomeric heptahelical glycoproteins that recognize a broad array of extracellular mediators including cationic amines, lipids, peptides, proteins, and sensory agents. Thus far, much attention has been given towards the comprehension of intracellular signaling mechanisms activated by cell membrane GPCRs, which convert extracellular hormonal stimuli into acute, non-genomic (e.g., hormone secretion, muscle contraction, and cell metabolism) and delayed, genomic biological responses (e.g., cell division, proliferation, and apoptosis). However, with respect to the latter response, there is compelling evidence for a novel intracrine mode of genomic regulation by GPCRs that implies either the endocytosis and nuclear translocation of peripheral-liganded GPCR and (or) the activation of nuclearly located GPCR by endogenously produced, nonsecreted ligands. A noteworthy example of the last scenario is given by heptahelical receptors that are activated by bioactive lipoids (e.g., PGE(2) and PAF), many of which may be formed from bilayer membranes including those of the nucleus. The experimental evidence for the nuclear localization and signalling of GPCRs will be reviewed. We will also discuss possible molecular mechanisms responsible for the atypical compartmentalization of GPCRs at the cell nucleus, along with their role in gene expression.