Stable expression of a secreted form of the mouse IFN-gamma receptor by rat cells.

The biologic effects of IFN-gamma are mediated through a receptor that is expressed in relatively low abundance on normal mammalian cells. As a consequence, investigations of the physicochemical and ligand-binding properties of the purified receptor have been limited. The work reported here characterizes a secreted form of the receptor for mouse IFN-gamma, made by deletion of the nucleotides that code for the anchoring domain from a cDNA that encodes the receptor binding protein and its related signal peptide. When transfected into rat XC cells, this construct produced up to approximately 1 mg/liter of a secreted protein that had the characteristics of the native receptor. Both the secreted protein and its mRNA were of sizes that were consistent with loss of the transmembrane region. The protein was detectable by a mAb that is specific for an epitope that is found in the ligand binding site of the receptor for mouse IFN-gamma, as well as by a goat polyclonal IgG that is monospecific for the mouse IFN-gamma R. Supernates that contained the secreted protein blocked binding of IFN-gamma to mouse IFN-gamma R and inhibited in a dose-dependent manner the IFN-gamma-mediated priming of mouse bone marrow culture-derived macrophages for tumor cell killing. Availability of relatively large amounts of a secreted protein that retains ligand-binding activity should facilitate purification and basic studies of the receptor binding protein and could provide new approaches to the treatment/prevention of diseases that arise due to inappropriate response of cells to IFN-gamma. In addition, because this secreted receptor, unlike others, consists of both the extracellular and intracellular domains, it is likely that it will be useful in determining how the cytoplasmic portion of the receptor is involved in receptor function.     

The structure of the nasal chemosensory system in squamate reptiles. 2. Lubricatory capacity of the vomeronasal organ

The vomeronasal organ is a poorly understood accessory olfactory organ, present in many tetrapods. In mammals, amphibians and lepidosaurian reptiles, it is an encapsulated structure with a central, fluid-filled lumen. The morphology of the lubricatory system of the vomeronasal organ (the source of this fluid) varies among classes, being either intrinsic (mammalian and caecilian amphibian vomeronasal glands) or extrinsic (anuran and urodele nasal glands). In the few squamate reptiles thus far examined, there are no submucosal vomeronasal glands. In this study, we examined the vomeronasal organs of several species of Australian squamates using histological, histochemical and ultrastructural techniques, with the goal of determining the morphology of the lubricatory system in the vomeronasal organ. Histochemically, the fluid within the vomeronasal organ of all squamates is mucoserous, though it is uncertain whether mucous and serous constituents constitute separate components. The vomeronasal organ produces few secretory granules intrinsically, implying an extrinsic source for the luminal fluid. Of three possible candidates, the Harderian gland is the most likely extrinsic source of this secretion.     

Purification and characterization of mouse alpha-lactalbumin from lactating mammary glands

The whey protein, alpha-lactalbumin, was purified from lactating mammary glands of mice at high yields. It exists as two major charge forms (pI values of 6.2 and 5.8) with similar molecular weights (approx. 14600). Antibodies prepared against these peptides precipitate newly synthesized and secreted alpha-lactalbumin from organ cultures of mid-pregnancy mammary glands. The antibody is specific for mouse alpha-lactalbumin as it does not react with mouse casein, mouse serum or purified bovine alpha-lactalbumin or galactosyl transferase. In addition, it blocks enzymatic activity of alpha-lactalbumin in mouse milk but has no effect on guinea pig or human milk. A very sensitive radioimmunoassay has been developed with this antibody which can detect alpha-lactalbumin levels as low as 0.25 ng.     

Morphological evidence for a direct innervation of the mouse vomeronasal glands

Summary The morphological evidence for a direct autonomic innervation of the mouse vomeronasal glands is presented. Axonal varicosities containing a few densecore vesicles and numerous clear vesicles (36–60 nm in diameter) make synaptic contacts with the secretory cells at the base of the glandular acini. The axonal presynaptic membrane is associated with a distinct dense material and it is separated from the secretory cell by a synaptic cleft of about 12–14 nm. At the postsynaptical level, coated vesicles can be found. Additional postsynaptical specializations have not been observed.     

Conditional knockout of TMEM16A/anoctamin1 abolishes the calcium-activated chloride current in mouse vomeronasal sensory neurons

Pheromones are substances released from animals that, when detected by the vomeronasal organ of other individuals of the same species, affect their physiology and behavior. Pheromone binding to receptors on microvilli on the dendritic knobs of vomeronasal sensory neurons activates a second messenger cascade to produce an increase in intracellular Ca²⁺ concentration. Here, we used whole-cell and inside-out patch-clamp analysis to provide a functional characterization of currents activated by Ca²⁺ in isolated mouse vomeronasal sensory neurons in the absence of intracellular K⁺. In whole-cell recordings, the average current in 1.5 µM Ca²⁺ and symmetrical Cl⁻ was −382 pA at −100 mV. Ion substitution experiments and partial blockade by commonly used Cl⁻ channel blockers indicated that Ca²⁺ activates mainly anionic currents in these neurons. Recordings from inside-out patches from dendritic knobs of mouse vomeronasal sensory neurons confirmed the presence of Ca²⁺-activated Cl⁻ channels in the knobs and/or microvilli. We compared the electrophysiological properties of the native currents with those mediated by heterologously expressed TMEM16A/anoctamin1 or TMEM16B/anoctamin2 Ca²⁺-activated Cl⁻ channels, which are coexpressed in microvilli of mouse vomeronasal sensory neurons, and found a closer resemblance to those of TMEM16A. We used the Cre–loxP system to selectively knock out TMEM16A in cells expressing the olfactory marker protein, which is found in mature vomeronasal sensory neurons. Immunohistochemistry confirmed the specific ablation of TMEM16A in vomeronasal neurons. Ca²⁺-activated currents were abolished in vomeronasal sensory neurons of TMEM16A conditional knockout mice, demonstrating that TMEM16A is an essential component of Ca²⁺-activated Cl⁻ currents in mouse vomeronasal sensory neurons.     

Purification and characterization of mouse α-lactalbumin from lactating mammary glands

The whey protein, α-lactalbumin, was purified from lactating mammary glands of mice at high yields. It exists as two major charge forms (pI values of 6.2 and 5.8) with similar molecular weights (approx. 14 00). Antibodies prepared against these peptides precipitate newly synthesized and secreted α-lactalbumin from organ cultures of mid-pregnancy mammary glands. The antibody is specific for mouse α-lactalbumin as it does not react with mouse casein, mouse serum or purified bovine α-lactalbumin or galactosyl transferase. In addition, it blocks enzymatic activity of α-lactalbumin in mouse milk but has no effect on guinea pig or human milk. A very sensitive radioimmunoassay has been developed with this antibody which can detect α-lactalbumin levels as low as 0.25 ng.     

Multiple forms of mouse vascular endothelial growth factor-D are generated by RNA splicing and proteolysis

The secreted glycoprotein vascular endothelial growth factor-D (VEGF-D) is angiogenic, lymphangiogenic, and promotes metastatic spread of tumor cells via lymphatic vessels. VEGF-D consists of a receptor-binding domain (VEGF homology domain) and N- and C-terminal propeptides. Proteolytic processing produces numerous forms of human VEGF-D, including fully processed derivatives (containing only the VEGF homology domain), partially processed, and unprocessed derivatives. Proteolysis is essential to generate human VEGF-D that binds the angiogenic receptor VEGF receptor-2 (VEGFR-2) and the lymphangiogenic receptor VEGFR-3 with high affinity. Here, we report that alternative use of an RNA splice donor site in exon 6 of the mouse VEGF-D gene produces two different protein isoforms, VEGF-D(358) and VEGF-D(326), with distinct C termini. The two isoforms were both expressed in all adult mouse tissues and embryonic stages of development analyzed. Both isoforms are proteolytically processed in a similar fashion to human VEGF-D to generate a range of secreted derivatives and bind and cross-link VEGFR-3 with similar potency. The isoforms are differently glycosylated when expressed in vitro. This study demonstrates that RNA splicing, protein glycosylation, and proteolysis are mechanisms for generating structural diversity of mouse VEGF-D.     

Calcium-activated chloride channels in the apical region of mouse vomeronasal sensory neurons

The rodent vomeronasal organ plays a crucial role in several social behaviors. Detection of pheromones or other emitted signaling molecules occurs in the dendritic microvilli of vomeronasal sensory neurons, where the binding of molecules to vomeronasal receptors leads to the influx of sodium and calcium ions mainly through the transient receptor potential canonical 2 (TRPC2) channel. To investigate the physiological role played by the increase in intracellular calcium concentration in the apical region of these neurons, we produced localized, rapid, and reproducible increases in calcium concentration with flash photolysis of caged calcium and measured calcium-activated currents with the whole cell voltage-clamp technique. On average, a large inward calcium-activated current of -261 pA was measured at -50 mV, rising with a time constant of 13 ms. Ion substitution experiments showed that this current is anion selective. Moreover, the chloride channel blockers niflumic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid partially inhibited the calcium-activated current. These results directly demonstrate that a large chloride current can be activated by calcium in the apical region of mouse vomeronasal sensory neurons. Furthermore, we showed by immunohistochemistry that the calcium-activated chloride channels TMEM16A/anoctamin1 and TMEM16B/anoctamin2 are present in the apical layer of the vomeronasal epithelium, where they largely colocalize with the TRPC2 transduction channel. Immunocytochemistry on isolated vomeronasal sensory neurons showed that TMEM16A and TMEM16B coexpress in the neuronal microvilli. Therefore, we conclude that microvilli of mouse vomeronasal sensory neurons have a high density of calcium-activated chloride channels that may play an important role in vomeronasal transduction.     

Identification of a functionally relevant signal peptide of mouse ficolin A

Mouse ficolin A is a plasma protein with lectin activity, and plays a role in host defense by binding carbohydrates, especially GlcNAc, on microorganisms. The ficolin A subunit consists of an N-terminal signal peptide, a collagen-like domain, and a C-terminal fibrinogen-like domain. In this study, we show that ficolin A can be synthesized and oligomerized in a cell and secreted into culture medium. We also identify a functionally relevant signal peptide of ficolin A by using MS/MS analysis to determine the N-terminal sequence of secreted ficolin A. When the signal peptide of mouse ficolin A was fused with enhanced green fluorescent protein (EGFP), EGFP was released into HEK 293 cell medium, suggesting that the signal peptide can efficiently direct ficolin A secretion. Moreover, our results suggest that the signal peptide of ficolin A has potential application for the production of useful secretory proteins.     

Mutation of a conserved hydrophobic patch prevents incorporation of ZP3 into the zona pellucida surrounding mouse eggs

Three glycoproteins (ZP1, ZP2, and ZP3) are synthesized in growing mouse oocytes and secreted to form an extracellular zona pellucida that mediates sperm binding and fertilization. Each has a signal peptide to direct it into a secretory pathway, a "zona" domain implicated in matrix polymerization and a transmembrane domain from which the ectodomain must be released. Using confocal microscopy and enhanced green fluorescent protein (EGFP), the intracellular trafficking of ZP3 was observed in growing mouse oocytes. Replacement of the zona domain with EGFP did not prevent secretion of ZP3, suggesting the presence of trafficking signals and a cleavage site in the carboxyl terminus. Analysis of linker-scanning mutations of a ZP3-EGFP fusion protein in transient assays and in transgenic mice identified an eight-amino-acid hydrophobic region required for secretion and incorporation into the zona pellucida. The hydrophobic patch is conserved among mouse zona proteins and lies between a potential proprotein convertase (furin) cleavage site and the transmembrane domain. The cleavage site that releases the ectodomain from the transmembrane domain was defined by mass spectrometry of native zonae pellucidae and lies N-terminal to a proprotein convertase site that is distinct from the hydrophobic patch.     

Chemical properties of a female mouse pheromone that stimulates gonadotropin secretion in males

Female mouse urine contains a pheromone that acts via the vomeronasal organ of conspecific males to stimulate a rapid increase in circulating levels of luteinizing hormone. A bioassay based on this male response was used to test biochemical preparations of female urine. Retention of significant biological activity by the urine after dialysis indicated that the activity is associated with urinary protein. Complete loss of activity from the urine after adsorption chromatography on a neutral polystyrene column suggested that the protein functions as a pheromone carrier. Assay of gel permeation chromatography fractions, before and after degradation of the urinary proteins with proteolytic enzymes, demonstrated that the protein is not necessary for the male response in the bioassay. Its resistance to vigorous proteolytic enzyme treatment further indicates that the pheromone is not a peptide. High biological activity, indistinguishable from that of the unfractionated urine, was isolated in a protein-depleted, presumably low molecular weight fraction containing compounds that are retarded by adsorption on Sephadex. The chemical properties of this female mouse pheromone are markedly different from those of a recently purified female hamster pheromone that also acts via the vomeronasal organ.     

Morphology of the nasal fossae and associated structures of the hamster (Mesocricetus auratus)

The hamster nasal cavity consists of vestibular, non-olfactory and olfactory portions. Much of the non-olfactory nasal cavity surface is lined by cuboidal, stratified cuboidal, and low columnar epithelia, devoid of cilia. Goblet cells and ciliated respiratory epithelium are present over only a small portion of the nasal cavity surface. The largest glandular masses in the hamster nose are the maxillary recess glands, the vomeronasal glands and the lateral nasal gland 1; these three glands contain neutral mucopolysaccharides (PAS-positive). Other nasal glands contain both acidic and neutral mucopolysaccharides; the staining reaction for acidic mucopolysaccharide is stronger in goblet cells and olfactory glands than in the other nasal glands. The ducts which open into the nasal vestibule are the excretory ducts of compound tubuloacinar serous glands. The one major PAS-positive gland whose duct opens into the nasal vestibule is the lateral nasal gland 1. The ducts of the compound tubuloacinar vomeronasal glands open into the lumen of the vomeronasal organ, which is connected to the ventral nasal meatus by means of the vomeronasal duct. The ducts of the branched tubuloacinar maxillary recess glands open into the maxillary recess. Few ducts open into the caudal half of the nasal cavity.     

Recombinant carboxyl-terminal fibrin-binding domain of human fibronectin expressed in mouse L cells

The carboxyl-terminal fibrin-binding domain, Fib2, of human fibronectin was expressed in mouse L cells as a fusion protein with the signal sequence of human protein C inhibitor. The recombinant Fib2 (rFib2) protein synthesized by transfected cells retained the ability to form dimers with each other or with mouse fibronectin subunits and was secreted to the medium after extensive glycosylation. Only a small fraction of the secreted protein was incorporated into the pericellular matrix. Interestingly, the secreted rFib2 protein displayed a remarkable heterogeneity upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, giving rise to a broad band corresponding to Mr of 60,000-90,000. The heterogeneity was eliminated mostly by treatment with neuraminidase and further by treatment with endo-alpha-N-acetylgalactosaminidase. Treatment with peptide:N-glycosidase F did not alter the heterogeneity of the protein, indicating that differential sialylation of O-linked, but not N-linked, glycans is largely responsible for the apparent heterogeneity. The presence of O-linked but absence of N-linked glycans was further supported by the observations that peanut agglutinin specifically bound to the desialylated rFib2 protein, whereas neither concanavalin A nor lentil lectin bound to the protein irrespective of prior neuraminidase treatment. Since the apparent heterogeneity of the rFib2 protein was only observable with the secreted, but not the cytoplasmic form, sialylation of O-linked glycans may be essential for, or regulate as a rate-limiting step, the transit of the recombinant protein to the extracellular space.     

Bile salt-stimulated lipase (BSSL) distribution in rat, mouse and transgenic mouse expressing human BSSL

 In some species, including man and mouse, bile salt-stimulated lipase (BSSL) in milk catalyzes the hydrolysis of triacylglycerides into glycerol and free fatty acids, a reaction that is of particular importance during suckling. The enzyme is also secreted by the pancreas (referred to as carboxyl-ester hydrolase, CEH). We wished to localize sources and storage sites for BSSL/CEH in rats, in wild-type mice, and in transgenic mice producing recombinant human BSSL in milk. Immunoreactivity against several BSSL fragments was strong in the pancreatic acinar cells and moderate in the absorptive cells of the small intestine and in salivary duct cells of the mice, as well as in rats. Sections from lactating mammary glands of mouse, but not rat, also showed immunoreactivity for BSSL; the signal was strongest in the transgenic mice. Radioactive riboprobes for BSSL mRNA hybridized on sections of rat and mouse pancreatic acinar cells, and mouse mammary glands (both wild-type and transgenic). Using RT-PCR, it was possible to amplify BSSL mRNA from wild-type mouse pancreas and mammary gland, from rat submandibular glands, and, in a few cases, from rat liver. In transgenic mice, the BSSL mRNA was highly expressed only in lactating mammary gland, but could be detected in a few other organs as well. Accepted: 31 March 1998     

Molecular cloning and functional characterization of mouse chitotriosidase

Mammalian chitinase and chitinase-like proteins are members of a recently discovered gene family. Thus far, neither chitin nor chitin synthase has been found in mammals. The existence of chitinase genes in mammals is intriguing and the physiologic functions of chitinases are not clear. Human chitotriosidase, also called chitinase 1 (chit1), has been cloned. It has been found that high levels of serum chitotriosidase are associated with several diseases, but the physiologic functions of this enzyme are still unclear. To facilitate the studies in animal models we cloned and characterized a cDNA that encodes the mouse chitotriosidase. The open reading frame of this cDNA predicts a protein of 464 amino acids with a typical chitinase structure, including a signal peptide, a highly conserved catalytic domain and a chitin-binding domain. The predicted amino acid sequence is highly homologous to that of human chitotriosidase and to that of mouse acidic mammalian chitinase. Sequence analysis indicates that the mouse chitotriosidase gene has 12 exons, spanning a 40-kb region in mouse chromosome 1. The constitutive expression of mouse chitotriosidase is restricted to brain, skin, bone marrow, kidney, tongue, stomach and testis. Recombinant expression of the cloned cDNA demonstrated that the encoded protein is secreted and has chitinolytic activity that is sensitive to the specific chitinase inhibitor allosamidin and has the ability to bind to chitin particles. Substitution mutations at the conserved catalytic site completely abolished the enzymatic activity of the recombinant protein. These studies illustrate that mouse chitotriosidase is a typical chitinase that belongs to the mammalian chitinase gene family.     

Electrophoretic isolation and partial characterization of a glycoprotein of the submandibular glands of the mouse

A protein has been isolated from the water-extract of the submandibular glands of the mouse, after Biogel P-300 column passage, followed by preparative polyacrylamide gel electrophoresis at pH 4.3 and subsequently at pH 8.9, designated the AM1 protein. The isolated protein was electrophoretically pure in 7.5, 10 and 15% polyacrylamide gels both at pH 4.3 and at pH 8.9. Likewise, by electrophoresis in 15% sodium dodecyl sulfate-polyacrylamide gel only one protein band could be detected. Of the total amount of the water-extractable submandibular proteins the recovery of this protein component comprised 3 to 5 per cent. The molecular weight was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 28 000, both in 7.5 and 15% gel. The isoelectric point was determined by isoelectric focusing in 4.8% polyacrylamide slabgel to be 4.85. The amino acid analysis showed that the ratio of acidic amino acids (Glx plus Asx) to basic amino acids (Lys plus Arg) is 2.3. The glycoprotein consists of protein for 77.4 per cent and of carbohydrate for 22.6 per cent. The molar ratio of the carbohydrates was GlcNH2:GalNH2:Man:Gal:Glc:Fuc:sialic acid = 22.0:1.3:3.0:1.7:10.0:2.6:0.3. The glycoprotein was not secreted from the submandibular glands by stimulation with cholinergic (acetylcholin) or adrenergic (noradrenalin) drugs both in vitro and in vivo. So, it appeared that this glycoprotein could be characterized as a cellular, non-secretory component of these salivary glands.     

Spatially and temporally regulated expression of specific heparan sulfate epitopes in the developing mouse olfactory system

Heparan sulfate (HS) comprises a structurally diverse group of glycosaminoglycans present ubiquitously on cell surfaces and in the extracellular matrix. The spatially and temporally regulated expression of specific HS structures is essential for various developmental processes in the nervous system but their distributions in the mouse olfactory system have not been explored. Here, we examined the spatiotemporal distribution of particular HS species in the developing mouse olfactory system using three structure‐specific monoclonal antibodies (HepSS‐1, JM403 and NAH46). The major findings were as follows. (i) During olfactory bulb morphogenesis, the HepSS‐1 epitope was strongly expressed in anterior telencephalic cells and coexpressed with fibroblast growth factor receptor 1. (ii) In early postnatal glomeruli, the JM403 epitope was expressed at different levels among individual glomeruli. The expression pattern and levels of the JM403 epitope were both associated with those of ephrin‐A3. (iii) In the vomeronasal system, the JM403 epitope was expressed in all vomeronasal axons but became increasingly restricted to vomeronasal axons terminating in the anterior region of the accessory olfactory bulb by 3 weeks of age. Our results demonstrate that each HS epitope exhibits a unique expression pattern during the development of the mouse olfactory system. Thus, each HS epitope is closely associated with particular developmental processes of the olfactory system and might have a particular role in developmental events.