A sulfotransferase-sulfatase system in avian oviduct which catalyzes a conversion of UDP-N-acetylgalactosamine 4-sulfate to the 6-sulfate isomer

Magnum from quail oviduct was subfractionated to yield epithelium and tubular glands. The in vitro enzymatic activities involved in sulfated sugar nucleotide biosynthesis were assayed in these isolated tissues. The results demonstrated that the activities necessary for a series of reactions, UDP-N-acetylgalactosamine----UDP-N-acetylgalactosamine 4-sulfate----UDP-N-acetylgalactosamine 4,6- bisulfate ----UDP-N-acetylgalactosamine 6-sulfate, are located predominantly in the tubular gland. Both time course and pulse-chase studies with [35S]sulfate gave results that were consistent with this reaction scheme. A microsomal preparation from the magnum was shown to be capable of labeling all three sulfate sugar nucleotides with [35S]sulfate upon incubation with UDP-N-acetylgalactosamine and 3'- phosphoadenylyl [35S]sulfate. Again, their relative labeling rates were in the order necessary to allow for a synthesis of sulfated sugar nucleotides in the sequence described above. Furthermore, incubation of the microsomal preparation with UDP-N-[14C]acetylgalactosamine 4-sulfate and 3'- phosphoadenylyl sulfate resulted in the formation of UDP-N-[14C]acetylgalactosamine 6-sulfate. Also shown was the existence in the microsomal preparation of a sulfatase specific for the sulfate at position 4 of UDP-N-acetylgalactosamine 4,6- bisulfate . The results, together with those obtained in previous investigations, suggest that the tubular gland of quail oviduct contains a microsomal multienzyme system which catalyzes a series of sulfation and desulfation of N-acetylgalactosamine residues at the nonreducing terminal position of either sugar nucleotides or polysaccharide chains.     

Profilin and the Abl tyrosine kinase are required for motor axon outgrowth in the Drosophila embryo

The ability of neuronal growth cones to be guided by extracellular cues requires intimate communication between signal transduction systems and the dynamic actin-based cytoskeleton at the leading edge. Profilin, a small, actin-binding protein, has been proposed to be a regulator of the cell motility machinery at leading edge membranes. However, its requirement in the developing nervous system has been unknown. Profilin associates with members of the Enabled family of proteins, suggesting that Profilin might link Abl function to the cytoskeleton. Here, genetic analysis in Drosophila is used to demonstrate that mutations in Profilin (chickadee) and Abl (abl) display an identical growth cone arrest phenotype for axons of intersegmental nerve b (ISNb). Moreover, the phenotype of a double mutant suggests that these components function together to control axonal outgrowth.     

Bacterial cholesterol oxidases are able to act as flavoprotein-linked ketosteroid monooxygenases that catalyse the hydroxylation of cholesterol to 4-cholesten-6-ol-3-one

A new metabolite of cholesterol was found in reaction mixtures containing cholesterol or 4-cholesten-3-one as a substrate and extra- or intracellular protein extracts from recombinant Streptomyces lividans and Escherichia coli strains carrying cloned DNA fragments of Streptomyces sp. SA-COO, the producer of Streptomyces cholesterol oxidase. The new metabolite was identified as 4-cholesten-6-ol-3-one based on comparisons of its high-performance liquid chromatography, gas chromatography/mass spectrometry, infrared and proton-nuclear magnetic resonance spectra with those of an authentic standard. Genetic analyses showed that the enzyme responsible for the production of 4-cholesten-6-ol-3-one is cholesterol oxidase encoded by the choA gene. Commercially purified cholesterol oxidase (EC 1.1.3.6.) of a Streptomyces sp., as well as of Brevibacterium sterolicum and a Pseudomonas sp., and a highly purified recombinant Streptomyces cholesterol oxidase were also able to catalyse the 6-hydroxylation reaction. Hydrogen peroxide accumulating in the reaction mixtures as a consequence of the 3β-hydroxysteroid oxidase activity of the enzyme was shown to have no role in the formation of the 6-hydroxylated derivative. We propose a possible scheme of a branched reaction pathway for the concurrent formation of 4-cholesten-3-one and 4-chotesten-6-ol-3-one by cholesterol oxidase, and the observed differences in the rate of formation of the 6-hydroxy-ketosteroid by the enzymes of different bacterial sources are also discussed.     

The core planar cell polarity gene prickle interacts with flamingo to promote sensory axon advance in the Drosophila embryo

The atypical cadherin Drosophila protein Flamingo and its vertebrate homologues play widespread roles in the regulation of both dendrite and axon growth. However, little is understood about the molecular mechanisms that underpin these functions. Whereas flamingo interacts with a well-defined group of genes in regulating planar cell polarity, previous studies have uncovered little evidence that the other core planar cell polarity genes are involved in regulation of neurite growth. We present data in this study showing that the planar cell polarity gene prickle interacts with flamingo in regulating sensory axon advance at a key choice point — the transition between the peripheral nervous system and the central nervous system. The cytoplasmic tail of the Flamingo protein is not required for this interaction. Overexpression of another core planar cell polarity gene dishevelled produces a similar phenotype to prickle mutants, suggesting that this gene may also play a role in regulation of sensory axon advance.     

N-acetylglucosamine 6-sulfate residues in keratan sulfate and heparan sulfate are desulfated by the same enzyme

We have prepared a series of oligosaccharides to assess the substrate specificity of exo sulfatase activity in cultured human skin fibroblasts toward N-acetylglucosamine-6-sulfate residues present in keratan sulfate (KS) and heparan sulfate (HS). Non-reducing end alpha-GlcNAc-6-SO4 residues (derived from HS) were desulfated by a specific sulfatase that when deficient leads to the accumulation of HS and the expression of mucopolysaccharidosis type IIID (Sanfilippo D). Under the in vitro conditions studied there are two pathways for the degradation of oligosaccharides containing non-reducing end beta-GlcNAc-6-SO4 residues (derived from KS). In one pathway beta-N-acetylglucosaminidase produces GlcNAc-6-SO4 which is then desulfated. In the other pathway the beta-GlcNAc-6-SO4 residue is desulfated and then cleaved by the action of an beta-N-acetylglucosaminidase activity. There was no detectable beta-N-acetylglucosaminidase activity in fibroblasts from a Tay-Sachs patient to produce GlcNAc-6-SO4 from beta-GlcNAc-6-SO4 residues in KS of oligosaccharides. There was approximately 10% of this normal beta-N-acetylglucosaminidase activity in fibroblasts from a Sandhoff patient, suggesting the A and S forms may be involved in this reaction. Desulfation of GlcNAc-6-SO4 residues in KS, HS and the monosaccharide GlcNAc-6-SO4 was considerably reduced or not detected in fibroblasts from a Sanfilippo D patient. As KS was not detected in the urine of a Sanfilippo D patient we propose that KS degradation in these patients proceeds by the action of a beta-N-acetylglucosaminidase activity to produce GlcNAc-6-SO4 which is not further degraded.(ABSTRACT TRUNCATED AT 250 WORDS)     

Block of muscle nicotinic receptors by choline suggests that the activation and desensitization gates act as distinct molecular entities

Ion channel block in muscle acetylcholine nicotinic receptors (AChRs) is an extensively reported phenomenon. Yet, the mechanisms underlying the interruption of ion flow or the interaction of the blocker with the channel's gates remain incompletely characterized. In this paper, we studied fast channel block by choline, a quaternary-ammonium cation that is also an endogenous weak agonist of this receptor, and a valuable tool in structure-function studies. Analysis of the single-channel current amplitude as a function of both choline concentration and voltage revealed that extracellular choline binds to the open-channel pore with millimolar apparent affinity (K(B) congruent with 12 mM in the presence of approximately 155 mM monovalent and 3.5 mM divalent, inorganic cations), and that it permeates the channel faster than acetylcholine. This, together with its relatively small size ( approximately 5.5 A along its longest axis), suggests that the pore-blocking choline binding site is the selectivity filter itself, and that current blockages simply reflect the longer-lived sojourns of choline at this site. Kinetic analysis of single-channel traces indicated that increasing occupancy of the pore-blocking site by choline (as judged from the reduction of the single-channel current amplitude) is accompanied by the lengthening of (apparent) open interval durations. Consideration of a number of possible mechanisms firmly suggests that this prolongation results from the local effect of choline interfering with the operation of the activation gate (closure of blocked receptors is slower than that of unblocked receptors by a factor of approximately 13), whereas closure of the desensitization gate remains unaffected. Thus, we suggest that these two gates act as distinct molecular entities. Also, the detailed understanding gained here on how choline distorts the observed open-time durations can be used to compensate for this artifact during activation assays. This correction is necessary if we are to understand how choline binds to and gates the AChR.     

Slitrk6 expression profile in the mouse embryo and its relationship to that of Nlrr3

Slitrk6 is a member of the Slitrk family of proteins, which are integral membrane proteins possessing two leucine-rich repeat (LRR) domains and a carboxy-terminal domain partially similar to that in the trk neurotrophin receptor proteins. Here, I show that Slitrk6 is uniquely expressed in various organs, different from other Slitrk genes which are predominantly expressed in neural tissues. In the developing mouse embryo, Slitrk6 expression was detected in the otic cyst, lateral trunk epidermis and its underlying mesenchymal tissue, limb bud, maxillary process, pharyngeal arches, cochlea, retina, tongue, tooth primordium, central nervous system (CNS), and the visceral organ primordia including of the lung, gastrointestinal tract (particularly in the enteric neurons) and pancreas. The expression in these organs occurred in a spatially restricted manner. In the CNS, the expression was highly compartmentalized in the dorsal thalamus, cerebellum and medulla. The expression compartment in the thalamus in which Slitrk6 was expressed was closely related to the Gbx2-expressing prosomere 2. Interestingly, the Slitrk6 expression in the CNS, cochlea, tongue, tooth primordial, and other organs was partially complementary to the expression of Nlrr3, which belongs to another family of neuronal LRR-containing transmembrane proteins. The complementary expression of the two proteins in the dorsal thalamus persisted from E13.5 to the adult stage.     

Roundabout gene family functions during sensory axon guidance in the drosophila embryo are mediated by both Slit-dependent and Slit-independent mechanisms

roundabout (robo) family genes play key roles in axon guidance in a wide variety of animals. We have investigated the roles of the robo family members, robo, robo2, and robo3, in the guidance of sensory axons in the Drosophila embryo. In robo(-/-), slit(-/-), and robo(-/+) slit(-/+) mutants, lateral cluster sensory neurons misproject to cells and axons in the nearby ventral' (v') cluster. These phenotypes, together with the normal expression pattern of Slit and Robo, suggest that Slit ligand secreted from the epidermis interacts with Robo receptors on lateral cluster sensory growth cones to limit their exploration of nearby attractive substrates. The most common sensory axon phenotype seen in robo2(-/-) mutants was misprojection of dorsal cluster sensory axons away from their normal growth substrate, the transverse connective of the trachea. slit appears to play no role in this aspect of sensory axon growth. Robo2 is expressed, not on the dorsal sensory axons, but on the transverse connective. These results suggest a novel, non-cell-autonomous mechanism for axon guidance by robo family genes: Robo2 expressed on the trachea acts as an attractant for the dorsal sensory growth cones.     

Characterization of complexes formed between TSG-6 and inter-alpha-inhibitor that act as intermediates in the covalent transfer of heavy chains onto hyaluronan

The high molecular mass glycosaminoglycan hyaluronan (HA) can become modified by the covalent attachment of heavy chains (HCs) derived from the serum protein inter-alpha-inhibitor (IalphaI), which is composed of three subunits (HC1, HC2 and bikunin) linked together via a chondroitin sulfate moiety. The formation of HC.HA is likely to play an important role in the stabilization of HA-rich extracellular matrices in the context of inflammatory disease (e.g. arthritis) and ovulation. Here, we have characterized the complexes formed in vitro between purified human IalphaI and recombinant human TSG-6 (an inflammation-associated protein implicated previously in this process) and show that these complexes (i.e. TSG-6 x HC1 and TSG-6 x HC2) act as intermediates in the formation of HC x HA. This is likely to involve two transesterification reactions in which an ester bond linking an HC to chondroitin sulfate in intact IalphaI is transferred first onto TSG-6 and then onto HA. The formation of TSG-6 x HC1 and TSG-6 x C2 complexes was accompanied by the production of bikunin x HC2 and bikunin x HC1 by-products, respectively, which were observed to break down, releasing free bikunin and HCs. Both TSG-6 x HC formation and the subsequent HC transfer are metal ion-dependent processes; these reactions have a requirement for either Mg2+ or Mn2+ and are inhibited by Co2+. TSG-6, which is released upon the transfer of HCs from TSG-6 onto HA, was shown to combine with IalphaI to form new TSG-6 x HC complexes and thus be recycled. The finding that TSG-6 acts as cofactor and catalyst in the production of HC x HA complexes has important implications for our understanding of inflammatory and inflammation-like processes.     

A chemotactic model for the advance and retreat of the primitive streak in avian development

The formation of the primitive streak in early avian development marks the onset of gastrulation, during which large scale cell movement leads to a trilaminar blastoderm comprising prospective endodermal, mesodermal and ectodermal tissue. During streak formation a specialized group of cells first moves anteriorly as a coherent column, beginning from the posterior end of the prospective anterior-posterior axis (a process called progression), and then reverses course and returns to the most posterior point on the axis (a process called regression). To date little is known concerning the mechanisms controlling either progression or regression. Here we develop a model in which chemotaxis directs the cell movement and which is capable of reproducing the principal features connected with progression and regression of the primitive streak. We show that this model exhibits a number of experimentally-observed features of normal and abnormal streak development, and we propose a number of experimental tests which may serve to illuminate the mechanisms. This paper represents the first attempt to model the global features of primitive streak formation, and provides an initial stage in the development of a more biologically-realistic discrete cell model that will allow for variation of properties between cells and control over movement of individual cells.     

DNA sequences homologous to the Drosophila opa repeat are present in murine mRNAs that are differentially expressed in fetuses and adult tissues.

A mouse embryonic cDNA containing two opa-like (CAX)n repeats was isolated on the basis of its cross-hybridization with a Drosophila K10 cDNA. Such repeated sequences were present in different murine mRNAs, some of which were specifically expressed during fetal life or in different adult tissues. This suggests that, as already described for Drosophila, opa-like sequences are parts of proteins involved in ontogenic or cell-type-specific functions in vertebrates. However, unlike Drosophila, such repeated sequences were not found within the murine homeo-boxes containing genes of the Hox-1 complex.     

Calnexin and BiP act as sequential molecular chaperones during thyroglobulin folding in the endoplasmic reticulum

Before secretion, newly synthesized thyroglobulin (Tg) folds via a series of intermediates: disulfide-linked aggregates and unfolded monomers-->folded monomers-->dimers. Immediately after synthesis, very little Tg associated with calnexin (a membrane-bound molecular chaperone in the ER), while a larger fraction bound BiP (a lumenal ER chaperone); dissociation from these chaperones showed superficially similar kinetics. Calnexin might bind selectively to carbohydrates within glycoproteins, or to hydrophobic surfaces of secretory proteins while they form proper disulfide bonds (Wada, I., W.-J. Ou, M.-C. Liu, and G. Scheele, J. Biol. Chem. 1994. 269:7464-7472). Because Tg has multiple disulfides, as well as glycans, we tested a brief exposure of live thyrocytes to dithiothreitol, which resulted in quantitative aggregation of nascent Tg, as analyzed by SDS-PAGE of cells lysed without further reduction. Cells lysed in the presence of dithiothreitol under non-denaturing conditions caused Tg aggregates to run as reduced monomers. For cells lysed either way, after in vivo reduction, Tg coprecipitated with calnexin. After washout of dithiothreitol, nascent Tg aggregates dissolved intracellularly and were secreted ultimately. 1 h after washout, > or = 92% of labeled Tg was found to dissociate from calnexin, while the fraction of labeled Tg bound to BiP rose from 0 to approximately 40%, demonstrating a "precursor-product" relationship. Whereas intralumenal reduction was essential for efficient Tg coprecipitation with calnexin, Tg glycosylation was not required. These data are among the first to demonstrate sequential chaperone function involved in conformational maturation of nascent secretory proteins within the ER.     

Evidence that L-glutamate can act as an exogenous signal to modulate root growth and branching in Arabidopsis thaliana

The roots of many plant species are known to use inorganic nitrogen, in the form of , as a cue to initiate localized root proliferation within nutrient-rich patches of soil. We report here that, at micromolar concentrations and in a genotype-dependent manner, exogenous l-glutamate is also able to elicit complex changes in Arabidopsis root development. l-Glutamate is perceived specifically at the primary root tip and inhibits mitotic activity in the root apical meristem, but does not interfere with lateral root initiation or outgrowth. Only some time after emergence do lateral roots acquire l-glutamate sensitivity, indicating that their ability to respond to l-glutamate is developmentally regulated. Comparisons between different Arabidopsis ecotypes revealed a remarkable degree of natural variation in l-glutamate sensitivity, with C24 being the most sensitive. The aux1-7 auxin transport mutant had reduced l-glutamate sensitivity, suggesting a possible interaction between l-glutamate and auxin signaling. Surprisingly, two loss-of-function mutants at the AXR1 locus (axr1-3 and axr1-12) were hypersensitive to l-glutamate. A pharmacological approach, using agonists and antagonists of mammalian ionotropic glutamate receptors, was unable to provide evidence of a role for their plant homologs in sensing exogenous glutamate. We discuss the mechanism of l-glutamate sensing and the possible ecological significance of the observed l-glutamate-elicited changes in root architecture.