Polygalacturonase inhibiting protein in plant cell walls

Polygalacturonase inhibiting protein (PGIP) is localized in plant cell walls and plays an important role both in pectic substance metabolism and in prevention of the penetration of phytopathogenic microorganisms. Apparently, PGIP is responsible for the specificity of cell--cell interactions during pollination or inoculation by fungi nonpathogenic for the particular plant. PGIPs from different plants share a basic common structure. They are rather thermostable glycoproteins enriched with leucine and contain about 20% carbohydrates; the molecular weight varies between 37-54 kD. The synthesis of PGIP is encoded by one gene, and its expression is stimulated by injury and fungal infection. The resistance of plant tissues to infection frequently correlates with PGIP expression and with inhibiting action on fungal PG. Thus, PGIP is believed to be useful for gene engineering to obtain transgenic plants resistant to fungal infection or retaining commercial value during storage.     

Nature and Patterns of Proteins during Cotton Seed Development

Patterns of accumulation and ontogenetic relationships among proteins of cotton (Gossypium hirsutum L.) seeds were examined between 10 days postanthesis and maturity (56 days). Total and extractable nitrogen contents were determined; alkali- and water-soluble proteins were assayed quantitatively and electrophoretically. Two alkali-soluble proteins present in the electrophoretogram of mature embryos first appeared at 21 days postanthesis; most of the final profile was established by 28 days. Except for minor changes centering around the 7th week of development, the pattern from 28 days to maturity was marked by intensification of bands. The quantity of water-soluble nitrogen increased through the first 21 days of development, then declined until 42 days, when it again began to increase; it reached its highest level at maturity. There was evidence of a high peptide content 7 weeks postanthesis.     

Characterization of cerebroside (monoglycosylceramide) from the sea anemone, Metridium senile. Identification of the major long-chain base as an unusual dienic base with a methyl branch at a double bond.

1. Cerebroside of the sea anemone, Metridium senile, has been isolated (0.6 mg/g dry tissue weight) and structurally characterized. 2. The structure was shown by mass spectrometry, NMR spectroscopy and degradative studies as beta-glucopyranosylceramide. The major fatty acids were 16 : 0 and 20 : 0 D-2-hydroxy fatty acids. The major base was a novel base, D-erythro-1,3-dihydroxy-2-amino-9-methyl-trans-4, trans-8-octadecadiene. 3. Some unusual fatty acids of marine origin are suggested to originate in this long-chain base by metabolic conversion. 4. The implication of the methyl branch position of the base on our current view of sphingolipid function in the plasma membrane is discussed.     

Installation of O-glycan sulfation capacities in human HEK293 cells for display of sulfated mucins

The human genome contains at least 35 genes that encode Golgi sulfotransferases that function in the secretory pathway, where they are involved in decorating glycosaminoglycans, glycolipids, and glycoproteins with sulfate groups. Although a number of important interactions by proteins such as selectins, galectins, and sialic acid–binding immunoglobulin-like lectins are thought to mainly rely on sulfated O-glycans, our insight into the sulfotransferases that modify these glycoproteins, and in particular GalNAc-type O-glycoproteins, is limited. Moreover, sulfated mucins appear to accumulate in respiratory diseases, arthritis, and cancer. To explore further the genetic and biosynthetic regulation of sulfated O-glycans, here we expanded a cell-based glycan array in the human embryonic kidney 293 (HEK293) cell line with sulfation capacities. We stably engineered O-glycan sulfation capacities in HEK293 cells by site-directed knockin of sulfotransferase genes in combination with knockout of genes to eliminate endogenous O-glycan branching (core2 synthase gene GCNT1) and/or sialylation capacities in order to provide simplified substrates (core1 Galβ1–3GalNAcα1–O-Ser/Thr) for the introduced sulfotransferases. Expression of the galactose 3-O-sulfotransferase 2 in HEK293 cells resulted in sulfation of core1 and core2 O-glycans, whereas expression of galactose 3-O-sulfotransferase 4 resulted in sulfation of core1 only. We used the engineered cell library to dissect the binding specificity of galectin-4 and confirmed binding to the 3-O-sulfo-core1 O-glycan. This is a first step toward expanding the emerging cell-based glycan arrays with the important sulfation modification for display and production of glycoconjugates with sulfated O-glycans.     

Eicosanoids as endogenous regulators of leptin release and lipolysis by mouse adipose tissue in primary culture

Prostaglandin E(2) (PGE(2)) stimulated leptin release over a 24-h incubation of mouse adipose tissue in primary culture. The maximal stimulation of leptin release was seen with 100 nm PGE(2). The role of endogenous eicosanoids in the regulation of lipolysis and leptin formation was examined in the presence of NS-398, a selective cyclooxygenase-2 inhibitor. NS-398 at a concentration of 5 microm enhanced lipolysis by 30% and lowered leptin release by 24%. This concentration of NS-398 almost completely inhibited PGE(2) formation. An inhibition of basal lipolysis by PGE(2) or N(6)-cyclopentyladenosine (CPA) was seen in the presence but not in the absence of NS-398. CPA, whose receptor, like that of PGE(2) inhibits cyclic AMP accumulation in adipose tissue, also enhanced leptin release. These data indicate that PGE2 can stimulate leptin release and suggest that endogenous eicosanoids affect both lipolysis and leptin formation by mouse adipose tissue.     

Enhanced pial arteriolar sensitivity to bioactive agents following exposure to endothelin-1

Effects of prior exposure of pial arterioles to endothelin-1 (ET-1) (10(-9) M) on the constriction induced by the by-products of hemolyzed blood (5-HT, LTC4, LPA, and thromboxane analog U-46619) were examined. Piglets (age: 1-3 d) anesthetized with a mixture of ketamine hydrochloride and acepromazine were implanted with cranial windows, and anesthesia was maintained with alpha-chloralose. Topical applications of the by-products of hemolyzed blood mildly constricted pial arterioles. Following prior exposure of the microvessels to ET-1, application of the by-products of hemolyzed blood produced significantly potentiated and long-lasting constrictions compared to the controls. In another experiment, pretreatment of pial arterioles with U-46619 (10(-8) M) also potentiated the constriction induced by ET-1. The constriction produced was fast and longer-lasting. Thus, these data show that by-products of hemolyzed blood, though not potent vasoconstrictors per se, potently constricted pial arterioles in the presence of ET-1. The same agents in the CSF can also potentiate constriction induced by ET-1. Hence, by-products of hemolyzed blood may play a significant role in the initiation and maintenance of cerebral arterial narrowing observed following intracranial bleeding.     

Fluorescent leukotriene B4: potential applications

Leukotriene B4 (LTB4) is a potent lipid mediator of inflammation that acts primarily via a seven-transmembrane-spanning, G-protein-coupled receptor denoted BLT1. Here, we describe the synthesis and characterization of fluorescent analogs of LTB4 that are easy to produce, inexpensive, and without the disadvantages of a radioligand. Fluorescent LTB4 is useful for labeling LTB4 receptors for which no antibodies are available and for performing one-step fluorescence polarization assays conducive to high-throughput screening. We found that orange and green fluorescent LTB4 were full agonists that activated the LTB4 receptor BLT1 with EC50 values of 68 and 40 nM, respectively (4.5 nM for unmodified LTB4). Flow cytometric measurements and confocal imaging showed that fluorescent LTB4 colocalized with BLT1. Fluorescence polarization measurements showed that orange fluorescent LTB4 bound to BLT1 with a Kd of 66 nM and that this binding could be displaced by unlabeled LTB4 and other BLT1-specific ligands. Fluorescent LTB4 analogs were also able to displace tritiated LTB4. Orange fluorescent LTB4 binding to enhanced green fluorescent protein-tagged BLT1 could be observed using fluorescence resonance energy transfer. In addition to being a useful alternative to radiolabeled LTB4, the unique properties of fluorescently labeled LTB4 allow a variety of detection technologies to be used.