Simultaneous apocrine and merocrine secretion in the rat coagulating gland

The coagulating gland of the rat synthesizes two prevalent secretory proteins (transglutaminase and 115 K) that are discharched in a different manner, one being secreted in an apocrine fashion (transglutaminase) and the other one in a merocrine way (115 K). Differences in the intra- cellular pathway and the release of either protein were studied using immunofluorescence on semithin sections, immunoelectron microscopy of preembedding-processed chopper sections and postembedding-processed ultrathin sections of rat coagulating gland. Immunohistochemical staining using an anti-transglutaminase antibody resulted in dense labeling of the cytoplasm of secretory cells and their apical blebs, whereas the cisternae of the rough endoplasmic reticulum and the Golgi apparatus were completely unlabeled. When, on the contrary, the anti-115 K antiserum was used, dense labeling of the cisternae of the rough endoplasmic reticulum, the Golgi apparatus, and the secretory granules was seen. Intraluminal secretion was also labeled, but the secretory blebs remained unlabeled. Our findings show that, in the coagulating gland of the male rat, the two secretory proteins studied are processed in parallel, but at completely different intracellular pathways. They are released via different extrusion mechanisms. Transglutaminase is synthesized outside the endoplasmic reticulum, reaches the apical cell pole by free flow in the cytoplasm, and is released via apocrine blebs, the membranes of which appear to be derived from the apical plasma membrane. The protein 115 K, on the other hand, follows the classic route, being synthesized within the cisternae of rough endoplasmic reticulum, subsequently glycosylated in the Golgi apparatus, and released in a merocrine fashion. The mutual exclusion of the two secretory pathways and the regulation of the alternative release mechanism are still unresolved issues.     

Benzoic acid glucosinolate esters and other glucosinolates from Arabidopsis thaliana

The spectacular recent progress in Arabidopsis thaliana molecular genetics furnishes outstanding tools for studying the formation and function of all metabolites in this cruciferous species. One of the major groups of secondary metabolites in A. thaliana is the glucosinolates. These hydrophilic, sulfur-rich glycosides appear to serve as defenses against some generalist herbivores and pathogens, and as feeding and oviposition stimulants to specialist herbivores. To help study their biosynthesis and role in plant-insect interactions, we wanted to determine the complete glucosinolate content of A. thaliana. In previous studies, 24 glucosinolates had been identified from ecotype Columbia. We reinvestigated Columbia as well as additional ecotypes and mutant lines, and identified 12 further glucosinolates, including five novel compounds. Structures were elucidated by MS and NMR spectroscopy of their desulfated derivatives, and by enzymatic cleavage of the attached ester moieties. Four of the novel glucosinolates are benzoate esters isolated from the seeds. In all but one of these compounds, esterification is on the glucose moiety rather than the side chain, a very unusual feature for glucosinolates. Among additional glucosinolates identified were the first non-chain elongated, methionine-derived glucosinolate from A. thaliana and the first compounds that appear to be derived from leucine.     

Inhibition of L- and P-selectin by a rationally synthesized novel core 2-like branched structure containing GalNAc-Lewisx and Neu5Acalpha2- 3Galbeta1-3GalNAc sequences

The selectins interact in important normal and pathological situations with certain sialylated, fucosylated glycoconjugate ligands containing sialyl Lewisx(Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)GlcN Ac). Much effort has gone into the synthesis of sialylated and sulfated Lewisxanalogs as competitive ligands for the selectins. Since the natural selectin ligands GlyCAM-1 and PSGL-1 carry sialyl Lewisxas part of a branched Core 2 O-linked structure, we recently synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(SE-3Galbeta1++ +-3)GalNAc1alphaOMe and found it to be a moderately superior ligand for L and P-selectin (Koenig et al. , Glycobiology 7, 79-93, 1997). Other studies have shown that sulfate esters can replace sialic acid in some selectin ligands (Yeun et al. , Biochemistry, 31, 9126-9131, 1992; Imai et al. , Nature, 361, 555, 1993). Based upon these observations, we hypothesized that Neu5Acalpha2-3Galbeta1-3GalNAc might have the capability of interacting with L- and P-selectin. To examine this hypothesis, we synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(Neu5Acalpha2++ +-3Galbeta1-3)- GalNAc alpha1-OB, which was found to be 2- to 3-fold better than sialyl Lexfor P and L selectin, respectively. We also report the synthesis of an unusual structure GalNAcbeta1-4(Fucalpha1- 3)GlcNAcbeta1-OMe (GalNAc- Lewisx-O-methyl glycoside), which also proved to be a better inhibitor of L- and P-selectin than sialyl Lewisx-OMe. Combining this with our knowledge of Core 2 branched structures, we have synthesized a molecule that is 5- to 6-fold better at inhibiting L- and P-selectin than sialyl Lewisx-OMe, By contrast to unbranched structures, substitution of a sulfate ester group for a sialic acid residue in such a molecule resulted in a considerable loss of inhibition ability. Thus, the combination of a sialic acid residue on the primary (beta1-3) arm, and a modified Lexunit on the branched (beta1-6) arm on an O-linked Core 2 structure generated a monovalent synthetic oliogosaccharide inhibitor superior to SLexfor both L- and P-selectin.      

Beneficial effect of fluorocarbon emulsion media on the function of neuromuscular preparations in vitro

The effects of liquid fluorocarbons as bathing media were determined by use of in vitro neuromuscular preparations. Rat hemidiaphragms were bathed in either oxygenated fluorocarbon (FC) emulsion or standard oxygenated Krebs solution. Contractile force in response to simple supramaximal nerve stimuli as well as to high frequency stimulation was greater, while twitch:tetanus ratio was smaller in FC emulsion. With such medium, post-tetanic potentiation of contraction was also more consistently observed. Indirectly stimulated diaphragms survived longer in FC emulsion. After cessation of oxygenation, oxygen tension (ρO(2)) of the medium declined more rapidly with Krebs than with FC emulsion; ρO(2) directly correlated with force of contraction. Similarly, in the chick biventer cervicis preparation, FC emulsion enhanced nerve-stimulated force of contraction; returning the preparation to standard Krebs solution reversed this phenomenon. Dose-resonse curves of muscle contraction in response to acetycholine and KCl administration were shifted upward during FC emulsion superfusion. Frequency of miniature endplate potentials was lower in FC emulsion than that observed in Krebs solution, measured from the same cell of the rat diaphragm. Resting membrane potentials were also greater in muscle cells sampled from FC emulsion-bathed preparations. These data suggest that FC emulsion is superior to standard Krebs solution as a bathing medium for in vitro neuromuscular preparations by virtue of the high solubility of oxygen in it.     

IN VIVO LABELLING OF ACETYL-ASPARTYL PEPTIDES IN MOUSE BRAIN FROM INTRACRANIALLY AND INTRAPERITONEALLY ADMINISTERED ACETYL-l-[U-14C] ASPARTATE

Abstract— Following intracranial and intraperitoneal injection of acetyl- l -[U-¹⁴C]aspartate into mice about 5% and 0.7% of the radioactivity, respectively, was recovered from the brain after 30 min.
On chromatographic separation of the cationic and anionic compounds on a Dowex 50 column, the former fraction contained about 60% of the radioactivity, predominantly as labelled aspartate and glutamate. The anionic compounds, containing 20% of the labelled compounds, were fractionated in several chromatographic systems and resolved into a great variety of labelled peptidic compounds of which five acetyl-[U¹⁴-C]aspartyl peptides, containing two to four amino acids, were purified. One of these, acetyl-aspartyl glutamine, has not previously been found in brain.     

Molecular characterization of polygalacturonases as grass pollen-specific marker allergens: expulsion from pollen via submicronic respirable particles

Grass pollen belong to the most important allergen sources involved in the elicitation of allergic asthma. We have isolated cDNAs coding for Bermuda grass (Cynodon dactylon) and timothy grass (Phleum pratense) pollen allergens, belonging to a family of pectin-degrading enzymes (i.e., polygalacturonases). The corresponding allergens, termed Cyn d 13 and Phl p 13, represent glycoproteins of approximately 42 kDa and isoelectric points of 7.5. rPhl p 13 was expressed in Escherichia coli and purified to homogeneity. Immunogold electron microscopy using rabbit anti-rPhl p 13 Abs demonstrated that in dry pollen group 13, allergens represent primarily intracellular proteins, whereas exposure of pollen to rainwater caused a massive release of cytoplasmic material containing submicronic particles of respirable size, which were coated with group 13 allergens. The latter may explain respiratory sensitization to group 13 allergens and represents a possible pathomechanism in the induction of asthma attacks after heavy rainfalls. rPhl p 13 was recognized by 36% of grass pollen allergic patients, showed IgE binding capacity comparable to natural Phl p 13, and induced specific and dose-dependent basophil histamine release. Epitope mapping studies localized major IgE epitopes to the C terminus of the molecule outside the highly conserved functional polygalacturonase domains. The latter result explains why rPhl p 13 contains grass pollen-specific IgE epitopes and may be used to diagnose genuine sensitization to grass pollen. Our finding that rabbit anti-rPhl p 13 Abs blocked patients' IgE binding to the allergen suggests that rPhl p 13 may be used for immunotherapy of sensitized patients.     

Effect of pretreatment of rubber material on its biodegradability by various rubber degrading bacteria

The effect of pretreatment of several cis-1,4-polyisoprene containing rubbers on their biodegradability was examined. Tests were carried out with six recently isolated and characterized rubber degrading bacteria belonging to the genera Gordonia (strains Kb2, Kd2 and VH2), Mycobacterium, Micromonospora and Pseudomonas. All strains were able to use natural rubber (NR) as well as NR latex gloves as sole carbon source. Extraction of NR latex gloves by organic solvents resulted in an enhancement of growth for three of the selected strains. On the other hand, growth of Gordonia sp. (strain Kb2 and Kd2), Mycobacterium fortuitum NF4 and Micromonospora aurantiaca W2b on synthetic cis-1,4-polyisoprene did only occur after removal of the antioxidants, that are usually added during manufacture to prevent aging of the materials. Detailed degradation studies performed with Gordonia sp. Kb2 revealed an enhanced mineralization of pretreated NR latex gloves and mineralization of purified natural rubber (NR), indicating the actual mineralization of cis-1,4-polyisoprene rubber constituent even after removal of non-rubber constituent that may act as co-metabolic substrate and support microbial growth. Further analysis by scanning electron microscopy (SEM) clearly demonstrated the enhanced colonization efficiency of these bacteria towards pretreated NR latex gloves. Colonization was additionally visualized by staining of overgrown NR latex gloves with Schiff's reagent, and the purple color produced in the area of degradation was an evidence for the accumulation of aldehydes containing oligomers. Further enhancement of latex gloves degradation could be achieved after successive replacement of mineral salts medium during cultivation. Thereby, a rapid disintegration of untreated NR latex gloves material was accomplished by Gordonia sp. strain VH2.     

Gene duplication in the diversification of secondary metabolism: tandem 2-oxoglutarate-dependent dioxygenases control glucosinolate biosynthesis in Arabidopsis

Secondary metabolites are a diverse set of plant compounds believed to have numerous functions in plant-environment interactions. The large chemical diversity of secondary metabolites undoubtedly arises from an equally diverse set of enzymes responsible for their biosynthesis. However, little is known about the evolution of enzymes involved in secondary metabolism. We are studying the biosynthesis of glucosinolates, a large group of secondary metabolites, in Arabidopsis to investigate the evolution of enzymes involved in secondary metabolism. Arabidopsis contains natural variations in the presence of methylsulfinylalkyl, alkenyl, and hydroxyalkyl glucosinolates. In this article, we report the identification of genes encoding two 2-oxoglutarate--dependent dioxygenases that are responsible for this variation. These genes, AOP2 and AOP3, which map to the same position on chromosome IV, result from an apparent gene duplication and control the conversion of methylsulfinylalkyl glucosinolate to either the alkenyl or the hydroxyalkyl form. By heterologous expression in Escherichia and the correlation of gene expression patterns to the glucosinolate phenotype, we show that AOP2 catalyzes the conversion of methylsulfinylalkyl glucosinolates to alkenyl glucosinolates. Conversely, AOP3 directs the formation of hydroxyalkyl glucosinolates from methylsulfinylalkyl glucosinolates. No ecotype coexpressed both genes. Furthermore, the absence of functional AOP2 and AOP3 leads to the accumulation of the precursor methylsulfinylalkyl glucosinolates. A third member of this gene family, AOP1, is present in at least two forms and found in all ecotypes examined. However, its catalytic role is still uncertain.