Characterisation and expression of SPLUNC2, the human orthologue of rodent parotid secretory protein

We recently described the Palate Lung Nasal Clone (PLUNC) family of proteins as an extended group of proteins expressed in the upper airways, nose and mouth. Little is known about these proteins, but they are secreted into the airway and nasal lining fluids and saliva where, due to their structural similarity with lipopolysaccharide-binding protein and bactericidal/permeability-increasing protein, they may play a role in the innate immune defence. We now describe the generation and characterisation of novel affinity-purified antibodies to SPLUNC2, and use them to determine the expression of this, the major salivary gland PLUNC. Western blotting showed that the antibodies identified a number of distinct protein bands in saliva, whilst immunohistochemical analysis demonstrated protein expression in serous cells of the major salivary glands and in the ductal lumens as well as in cells of minor mucosal glands. Antibodies directed against distinct epitopes of the protein yielded different staining patterns in both minor and major salivary glands. Using RT-PCR of tissues from the oral cavity, coupled with EST analysis, we showed that the gene undergoes alternative splicing using two 5' non-coding exons, suggesting that the gene is regulated by alternative promoters. Comprehensive RACE analysis using salivary gland RNA as template failed to identify any additional exons. Analysis of saliva showed that SPLUNC2 is subject to N-glycosylation. Thus, our study shows that multiple SPLUNC2 isoforms are found in the oral cavity and suggest that these proteins may be differentially regulated in distinct tissues where they may function in the innate immune response

Base-Controlled Diastereoselective Synthesis of Either anti- or syn-β-Aminonitriles

Deprotonation of secondary alkane nitriles with nBuLi and addition to aryl imines gives kinetic anti-β-aminonitriles. Use of LHMDS allows reversible protonation of the reaction intermediate to give syn-β-aminonitriles. The pure diastereosiomers can be isolated in good yields, and the mechanism was elucidated

BPIFB1 (LPLUNC1) is upregulated in cystic fibrosis lung disease

Although the biology the PLUNC (recently renamed BPI fold, BPIF) family of secreted proteins is poorly understood, multiple array based studies have suggested that some are differentially expressed in lung diseases. We have examined the expression of BPIFB1 (LPLUNC1), the prototypic two-domain containing family member, in lungs from CF patients and in mouse models of CF lung disease. BPIFB1 was localized in CF lung samples along with BPIFA1, MUC5AC, CD68 and NE and directly compared to histologically normal lung tissues and that of bacterial pneumonia. We generated novel antibodies to mouse BPIF proteins to conduct similar studies on ENaC transgenic (ENaC-Tg) mice, a model for CF-like lung disease. Small airways in CF demonstrated marked epithelial staining of BPIFB1 in goblet cells but staining was absent from alveolar regions. BPIFA1 and BPIFB1 were not co-localised in the diseased lungs. In ENaC-Tg mice there was strong staining of both proteins in the airways and luminal contents. This was most marked for BPIFB1 and was noted within 2 weeks of birth. The two proteins were present in distinct cells within epithelium. BPIFB1 was readily detected in BAL from ENaC-Tg mice but was absent from wild-type mice. Alterations in the expression of BPIF proteins is associated with CF lung disease in humans and mice. It is unclear if this elevation of protein production, which results from phenotypic alteration of the cells within the diseased epithelium, plays a role in the pathogenesis of the disease

BPI-fold (BPIF) containing/plunc protein expression in human fetal major and minor salivary glands.

The aim of this study was to determine expression, not previously described, of PLUNC (palate, lung, and nasal epithelium clone) (BPI-fold containing) proteins in major and minor salivary glands from very early fetal tissue to the end of the second trimester and thus gain further insight into the function of these proteins. Early fetal heads, and major and minor salivary glands were collected retrospectively and glands were classified according to morphodifferentiation stage. Expression of BPI-fold containing proteins was localized through immunohistochemistry. BPIFA2, the major BPI-fold containing protein in adult salivary glands, was detected only in the laryngeal pharynx; the lack of staining in salivary glands suggested salivary expression is either very late in development or is only in adult tissues. Early expression of BPIFA1 was seen in the trachea and nasal cavity with salivary gland expression only seen in late morphodifferentiation stages. BPIFB1 was seen in early neural tissue and at later stages in submandibular and sublingual glands. BPIFA1 is significantly expressed in early fetal oral tissue but BPIFB1 has extremely limited expression and the major salivary BPIF protein (BPIFA2) is not produced in fetal development. Further studies, with more sensitive techniques, will confirm the expression pattern and enable a better understanding of embryonic BPIF protein function

Reductive conjugate addition nitro-Mannich route for the stereoselective synthesis of 1,2,3,4-tetrahydroquinoxalines

A concise, high yielding and structurally divergent synthesis of complex 1,2,3,4-tetrahydroquinoxalines with excellent diastereoselectivity is described. A wide array of nitroalkenes and imines derived from commercially available aromatic aldehydes and 2-chloroanalines were subjected to a key reductive conjugate addition nitro-Mannich reaction to give diastereomerically pure β-nitro amines. Sequential reduction of the nitro function followed by Pd-catalyzed intramolecular N-arylation of the resultant primary amine onto the 2-chloroanailine gives highly substituted 1,2,3,4-tetrahydroquinoxalines. Non basic imines were found to participate better in the nitro-Mannich reaction if the stronger acid methanesulfonic acid was used to promote the reaction. The 3 step reaction sequence should be useful for the array synthesis of drug like scaffolds

PhOTO Zebrafish: A Transgenic Resource for In Vivo Lineage Tracing during Development and Regeneration

Background: Elucidating the complex cell dynamics (divisions, movement, morphological changes, etc.) underlying embryonic development and adult tissue regeneration requires an efficient means to track cells with high fidelity in space and time. To satisfy this criterion, we developed a transgenic zebrafish line, called PhOTO, that allows photoconvertible optical tracking of nuclear and membrane dynamics in vivo. Methodology: PhOTO zebrafish ubiquitously express targeted blue fluorescent protein (FP) Cerulean and photoconvertible FP Dendra2 fusions, allowing for instantaneous, precise targeting and tracking of any number of cells using Dendra2 photoconversion while simultaneously monitoring global cell behavior and morphology. Expression persists through adulthood, making the PhOTO zebrafish an excellent tool for studying tissue regeneration: after tail fin amputation and photoconversion of a ~100µm stripe along the cut area, marked differences seen in how cells contribute to the new tissue give detailed insight into the dynamic process of regeneration. Photoconverted cells that contributed to the regenerate were separated into three distinct populations corresponding to the extent of cell division 7 days after amputation, and a subset of cells that divided the least were organized into an evenly spaced, linear orientation along the length of the newly regenerating fin. Conclusions/Significance: PhOTO zebrafish have wide applicability for lineage tracing at the systems-level in the early embryo as well as in the adult, making them ideal candidate tools for future research in development, traumatic injury and regeneration, cancer progression, and stem cell behavior

Bilinear R-parity violation with flavor symmetry

Bilinear R-parity violation (BRPV) provides the simplest intrinsically supersymmetric neutrino mass generation scheme. While neutrino mixing parameters can be probed in high energy accelerators, they are unfortunately not predicted by the theory. Here we propose a model based on the discrete flavor symmetry $A_4$ with a single R-parity violating parameter, leading to (i) correct Cabbibo mixing given by the Gatto-Sartori-Tonin formula, and a successful unification-like b-tau mass relation, and (ii) a correlation between the lepton mixing angles $\theta_{13}$ and $\theta_{23}$ in agreement with recent neutrino oscillation data, as well as a (nearly) massless neutrino, leading to absence of neutrinoless double beta decay.Comment: 16 pages, 3 figures. Extended version, as published in JHE