Characterisation of the human plunc gene, a gene product with an upper airways and nasopharyngeal restricted expression pattern

Here we report the cloning and characterization of the human homologue of plunc, a murine gene expressed specifically in the upper airways and nasopharyngeal regions. The human plunc cDNA codes for a leucine-rich protein of 256 amino acids which is 72% identical to the murine protein. RNA blot analysis suggests that expression of plunc is restricted to the trachea, upper airway, nasopharyngeal epithelium and salivary gland. The human plunc gene contains nine exons and is localised to chromosome 20q11.2. The unique expression pattern of the human plunc suggest that it may prove a useful model gene with which to study the regulatory mechanisms which direct expression of genes specifically to the upper airways.     

Cell-specific expression of CYP2A5 in the mouse respiratory tract: effects of olfactory toxicants.

We performed a detailed analysis of mouse cytochrome P450 2A5 (CYP2A5) expression by in situ hybridization (ISH) and immunohistochemistry (IHC) in the respiratory tissues of mice. The CYP2A5 mRNA and the corresponding protein co-localized at most sites and were predominantly detected in the olfactory region, with an expression in sustentacular cells, Bowman's gland, and duct cells. In the respiratory and transitional epithelium there was no or only weak expression. The nasolacrimal duct and the excretory ducts of nasal and salivary glands displayed expression, whereas no expression occurred in the acini. There was decreasing expression along the epithelial linings of the trachea and lower respiratory tract, whereas no expression occurred in the alveoli. The hepatic CYP2A5 inducers pyrazole and phenobarbital neither changed the CYP2A5 expression pattern nor damaged the olfactory mucosa. In contrast, the olfactory toxicants dichlobenil and methimazole induced characteristic changes. The damaged Bowman's glands displayed no expression, whereas the damaged epithelium expressed the enzyme. The CYP2A5 expression pattern is in accordance with previously reported localization of protein and DNA adducts and the toxicity of some CYP2A5 substrates. This suggests that CYP2A5 is an important determinant for the susceptibility of the nasal and respiratory epithelia to protoxicants and procarcinogens.     

The BSP30 salivary proteins from cattle,LUNX/PLUNC and von Ebner's minor salivary gland protein are members of the PSP/LBP superfamily of proteins

Saliva influences rumen function in cattle, yet the biochemical role for most of the bovine salivary proteins (BSPs) has yet to be established. Two cDNAs (BSP30a and BSP30b) from bovine parotid salivary gland were cloned and sequenced, each coding for alternate forms of a prominent protein in bovine saliva. The BSP30 cDNAs share 96% sequence identity with each other at the DNA level and 83% at the amino acid level, and appear to arise from separate genes. The predicted BSP30a and BSP30b proteins share 26-36% amino acid identity with parotid secretory protein (PSP) from mouse, rat and human. BSP30 and PSP are in turn more distantly related to a wider group of proteins that includes lung-specific X protein, also known as palate, lung, and nasal epithelium clone (LUNX/PLUNC), von Ebner's minor salivary gland protein (VEMSGP), bactericidal permeability increasing protein (BPI), lipopolysaccharide binding protein (LBP), cholesteryl ester transfer protein (CETP), and the putative olfactory ligand-binding proteins RYA3 and RY2G5. Bovine cDNAs encoding homologs of LUNX/PLUNC and VEMSGP were isolated and sequenced. Northern blot analysis showed that LUNX/PLUNC, BSP30 and VEMSGP are expressed in bovine salivary tissue and airways, and that they have non-identical patterns of expression in these tissues. The expression of both BSP30a and BSP30b is restricted to salivary tissue, but within this tissue they have distinct patterns of expression. The proximity of the human genes coding for the PSP/LBP superfamily on HSA20q11.2, their similar amino acid sequence, and common exon segmentation strongly suggest that these genes evolved from a common ancestral gene. Furthermore, they imply that the BSP30a and BSP30b proteins may have a function in common with other members of this gene family.     

扬子鳄鼻腔上皮光学显微镜及扫描电镜观察

本文通过光镜和扫描电镜研究了爬行动物扬子鳄鼻腔上皮的组织学。结果表明:其嗅觉上皮的组成细胞类型与两栖类、鸟类和哺乳类基本相似,但嗅细胞纤毛形状则有所不同;扬子鳄与两栖类、鸟类嗅纤毛相似,呈丝状,而哺乳类嗅觉纤毛则呈棍棒状;据外,扬子鳄鼻腔不同部位可发现不同类型嗅纤毛,鸟兽则无此现象,扬子鳄嗅觉上皮的分布仅局限于鼻腔中部前甲区和鼻甲区狭小范围,而兽类嗅觉上皮一般分布较广;扬子鳄呼吸上皮下未见兽类具有的混合型粘液腺,也未见兽类用以温暖空气的静脉丛,这和扬子鳄属外温动物而兽类为恒温动物密切相关。     

Cloning and expression of a mouse member of the PLUNC protein family exclusively expressed in tongue epithelium

Palate, lung, and nasal epithelium clone (Plunc, now renamed Splunc1) is a small secreted protein expressed in the oropharynx and upper airways of humans, mice, rats, and cows. This protein is structurally homologous to known mediators of host defense against gram-negative bacteria. We have characterized the genomic sequence and expression of a novel but closely related gene from rodents, which we call splunc5. Mouse Splunc5 sequence is 60% identical to mouse Splunc1. The genes also share highly conserved genomic elements including intron-exon structure and intronic sequence. Strikingly, splunc5 is expressed exclusively in the interpapillary epithelium of the tongue's dorsal surface. By comparing the expression profiles of splunc5, splunc1, and a third related sequence, lplunc1, in mice, we show that these genes are expressed in unique domains along a continuous corridor of oral, lingual, pharyngeal, and respiratory epithelia. This expression pattern is consistent with the hypothesis that these proteins protect epithelial surfaces colonized by potentially pathogenic microorganisms.     

Cathepsin Q, a novel lysosomal cysteine protease highly expressed in placenta

The complete nucleotide sequence of a novel cathepsin cDNA derived from rat placenta was determined and is termed cathepsin Q. The predicted protein of 343 amino acid is a member of the family C1A protease related to cathepsin L. Rat cathepsin Q and its mouse counterpart were found highly expressed in placenta, whereas no detectable levels were found in lung, spleen, heart, brain, kidney, thymus, testicle, liver, or embryonic tissues. It is predicted that cathepsin Q will differ in catalytic specificity to another placental-specific protease, cathepsin P, indicating that these enzymes will have unique proteolytic functions in extra-embryonic tissues.     

新的天然免疫保护分子——PLUNC家族蛋白

在呼吸道上皮与消化道上皮的表面,覆盖有一层由免疫保护分子所组成的蛋白质混合物,在上皮组织与外界各种信号之间,它们起着信号传递中介与信号执行分子的作用.我们新克隆的NASG基因为这一混合物添加了新的成员,对其结构与功能分析表明:它属于腭、肺及鼻咽上皮克隆（PLUNC）家族中SPLUNC1的全新转录本.目前发现人类PLUNC家族至少有8个以上成员,分布在人类20号染色体大约300 kb的狭窄区域,它们具有杀菌\渗透增强蛋白结构域,在进化上高度保守,每个成员分别在呼吸道上皮的不同部位特异性表达,具有潜在的结合细菌脂多糖的功能,能对外来物理及化学刺激做出反应,以分泌蛋白的形式进入鼻咽分泌物或唾液中,部分家族成员可能具有抗微生物、清除有害化学物质、抗肿瘤等多重功效.以上说明,PLUNC家族可能是上呼吸道的一种新的天然免疫保护分子,在维持上呼吸道的正常生理活动中起重要作用.     

Identification and biochemical analysis of novel olfactory-specific cytochrome P-450IIA and UDP-glucuronosyl transferase

Two major transmembranal polypeptides of bovine olfactory epithelium were identified by SDS electrophoretic analysis of Triton X-114 solubilized membranes. Both polypeptides were present in large amounts in membranes of the olfactory epithelium but were barely detectable in membranes of the nasal respiratory epithelium. Both polypeptides are enriched in the deciliated epithelium as compared with isolated cilia. One of them is a glycoprotein with an apparent molecular mass of 56 kDa (gp56); the other is an unglycosylated protein with an apparent molecular mass of 52 kDa (p52). Sequence analysis of peptides obtained by CNBr cleavage of purified gp56 indicates that it is highly homologous to UDP-glucuronosyl transferase (UDPGT). Parallel analysis shows that p52 is highly homologous to cytochrome P-450 sequences of the IIA subfamily. This protein is assigned the name P-450olf2. Polyclonal antibodies were raised against synthetic peptides corresponding to gp56 and p52 peptide sequences. Immunoblots with these antibodies reveal the following properties of gp56 and p52: (1) they are enriched in the microsomal fraction of the bovine olfactory epithelium; (2) they are possibly specific to the olfactory epithelium, as we could not detect reactivity in microsomes derived from respiratory epithelium or lung, and only a very small amount of basal reactivity was seen with liver microsomes; (3) cross-reacting proteins exist in microsomes derived from the rat olfactory epithelium. These results are consistent with a mechanism whereby the microsomal enzymes are involved in odorant modification and clearance from the nasal tissue.     

Carbonic anhydrase VI in the mouse nasal gland.

Western blotting analysis of mouse nasal tissue using a specific anti-mouse secreted carbonic anhydrase (CA VI) antibody has shown that CA VI is present in this tissue. A single immunoreactive band of 42 kD was observed, as has been found previously for salivary tissues. RT-PCR analysis has shown that nasal mucosa expressed CA VI mRNA. By immunohistochemistry (IHC), CA VI was observed in acinar cells, in duct contents of the anterior gland of the nasal septum, and in the lateral nasal gland. The Bowman's gland, the posterior gland of the nasal septum, and the maxillary sinus gland were negative. Immunoreactivity was also observed in the mucus covering the respiratory and olfactory mucosa and in the lumen of the nasolacrimal duct. In contrast, an anti-rat CA II antibody (that crossreacts with the mouse enzyme) stained only known CA II-positive cells and an occasional olfactory receptor neuron. These results indicate that CA VI is produced by the nasal gland and is secreted over the nasal mucosa. By reversible hydration of CO(2), CA VI is presumed to play a role in mucosal functions such as CO(2) sensation and acid-base balance. It may also play a role in olfactory function as a growth factor in maturation of the olfactory epithelial cells.     

Identification of a third rat odorant-binding protein (OBP3)

From a rat olfactory epithelium cDNA library clones encoding a lipocalin were isolated with sequence identity to the previously described salivary-specific alpha-2u globulin and the N-terminal region of mouse odorant-binding proteins OBP-III and OBP-IV. In situ hybridization showed strong expression in nasal glands displaying a pattern equivalent to rat OBP1. Heterologously expressed protein was evaluated for its binding properties using spectroscopic approaches. The recombinant protein interacted with two fluorescent probes, 1-aminoanthracene (1-AMA) and 1,1'-bis(4-anilino-5-naphthalene)-sulfonic acid. 1-AMA binding was competed by several odorants with high affinity. The thermodynamic parameters of the protein-odorant interaction were determined using isothermal titration calorimetry. Due to its nasal expression and odorant-binding characteristics this protein was designated OBP3.     

Antioxidant status of the rat nasal cavity

Despite extensive interest in the rodent nasal cavity as a target organ for toxicity, there is very limited information regarding nasal defenses against oxidative stress and xenobiotic-derived oxidants. Using immunohistochemistry, we have examined the distribution of Cu,Zn and Mn superoxide dismutase (SOD), catalase, glutathione (GSH) peroxidase, and DT-diaphorase in rat nasal tissues. In addition, we have determined the concentrations of ascorbate and alpha-tocopherol and the activities of SOD (combined Cu,Zn and Mn forms), catalase, GSH peroxidase, GSH reductase, and DT-diaphorase in nasal respiratory epithelium (RE), olfactory epithelium (OE), and in lung. Immunohistochemistry demonstrated that all four enzymes were similarly distributed, with the greatest staining intensity in dorsal-medial regions of the nasal cavity. In respiratory epithelium, ciliated columnar cells and subepithelial glands stained positively, while in olfactory tissue the enzymes were detected in the sustentacular cells and Bowman's glands. With the exception of SOD, enzyme activities were higher in RE than OE, while concentrations of ascorbate and alpha-tocopherol were higher in OE than RE. With the exception of catalase, nasal activities were either higher than or comparable to those of the lung. Thus, the rat nasal cavity appears to be well protected against oxidative damage.     

Antisera specific for the alpha 1, alpha 2, alpha 3, and beta subunits of the Na,K-ATPase: differential expression of alpha and beta subunits in rat tissue membranes

We have developed a panel of antibodies specific for the alpha 1, alpha 2, alpha 3, and beta subunits of the rat Na,K-ATPase. TrpE-alpha subunit isoform fusion proteins were used to generate three antisera, each of which reacted specifically with a distinct alpha subunit isotype. Western blot analysis of rat tissue microsomes revealed that alpha 1 subunits were expressed in all tissues while alpha 2 subunits were expressed in brain, heart, and lung. The alpha 3 subunit, a protein whose existence had been inferred from cDNA cloning, was expressed primarily in brain and copurified with ouabain-inhibitable Na,K-ATPase activity. An antiserum specific for the rat Na,K-ATPase beta subunit was generated from a TrpE-beta subunit fusion protein. Western blot analysis showed that beta subunits were present in kidney, brain, and heart. However, no beta subunits were detected in liver, lung, spleen, thymus, or lactating mammary gland. The distinct tissue distributions of alpha and beta subunits suggest that different members of the Na,K-ATPase family may have specialized functions.     

Immunolocalisation of P2X and P2Y nucleotide receptors in the rat nasal mucosa

Purinoceptor subtypes were localised to various tissue types present within the nasal cavity of the rat, using immunohistochemical methods. P2X₃ receptor immunoreactivity was localised in the primary olfactory neurones located both in the olfactory epithelium and vomeronasal organs (VNO) and also on subepithelial nerve fibres in the respiratory region. P2X₅ receptor immunoreactivity was found in the squamous, respiratory and olfactory epithelial cells of the rat nasal mucosa. P2X₇ receptor immunoreactivity was also expressed in epithelial cells and colocalised with caspase 9 (an apoptotic marker), suggesting an association with apoptosis and epithelial turnover. P2Y₁ receptor immunoreactivity was found within the respiratory epithelium and submucosal glandular tissue. P2Y₂ receptor immunoreactivity was localised to the mucus-secreting cells within the VNO. The possible functional roles of these receptors are discussed.     

Differential localisation of BPIFA1 (SPLUNC1) and BPIFB1 (LPLUNC1) in the nasal and oral cavities of mice

Despite being initially identified in mice, little is known about the sites of production of members of the BPI fold (BPIF) containing (PLUNC) family of putative innate defence proteins in this species. These proteins have largely been considered to be specificaly expressed in the respiratory tract, and we have recently shown that they exhibit differential expression in the epithelium of the proximal airways. In this study, we have used species-specific antibodies to systematically localize two members of this protein family; BPIFA1 (PLUNC/SPLUNC1) and BPIFB1 (LPLUNC1) in adult mice. In general, these proteins exhibit distinct and only partially overlapping localization. BPIFA1 is highly expressed in the respiratory epithelium and Bowman??s glands of the nasal passages, whereas BPIFB1 is present in small subset of goblet cells in the nasal passage and pharynx. BPIFB1 is also present in the serous glands in the proximal tongue where is co-localised with the salivary gland specific family member, BPIFA2E (parotid secretory protein) and also in glands of the soft palate. Both proteins exhibit limited expression outside of these regions. These results are consistent with the localization of the proteins seen in man. Knowledge of the complex expression patterns of BPIF proteins in these regions will allow the use of tractable mouse models of disease to dissect their function.     

Cloning, structural organization and chromosomal mapping of rat costimulatory molecule 4-1BBL

4-1BBL （TNFSF9） is a member of the tumor necrosis factor （TNF） ligand superfamily, which is expressed on some activated antigen presenting cells and B cells. We isolated a rat cDNA clone encoding the rat homologue of the human 4-1BBL （GenBank accession No. AY259541）. The deduced rat 4-1BBL protein, consisting of 308 amino acids with a molecular weight of 33,469 Da, was a typical type II transmembrane glycoprotein, the same as human and murine 4-1BBL. “SDAA” in the cytoplasmic domain of rat 4-1BBL was deduced to act as the phosphorylation site for casein kinase I （“SXXS” motif）, which is present in the cytoplasmic domains of human and murine 4-1BBL, and all other TNF ligand family members known to utilize reverse signaling. The two introns of 4-1BBL were also cloned （GenBank accession No. AY332409）. Rat 4-1BBL is much more homologous with murine 4-1BBL than with human 4-1BBL, in both nucleotide and amino acid sequences. Rat 4-1BBL was expressed in all tested tissues： brain, lung, colon, liver, thymus, testicle, kidney, adrenal, stomach, spleen and heart. The chromosomal location of rat 4-1BBL was first identified by bioinformatics, then by fluorescence in situ hybridization at 9q11 （GenBank accession UniGene No. Rn.46783）. Rat, murine and human 4-1BBL genes are evolved from a common gene. The identification and characterization of the rat counterpart of human 4-1BBL will facilitate studies of the biological function of this molecule.