Presence of the M-type sPLA(2) receptor on neutrophils and its role in elastase release and adhesion

Secretory phospholipase A(2) (sPLA(2)) produces lipids that stimulate polymorphonuclear neutrophils (PMNs). With the discovery of sPLA(2) receptors (sPLA(2)-R), we hypothesize that sPLA(2) stimulates PMNs through a receptor. Scatchard analysis was used to determine the presence of a sPLA(2) ligand. Lysates were probed with an antibody to the M-type sPLA(2)-R, and the immunoreactivity was localized. PMNs were treated with active and inactive (+EGTA) sPLA(2) (1-100 units of enzyme activity/ml, types IA, IB, and IIA), and elastase release and PMN adhesion were measured. PMNs incubated with inactive, FITC-linked sPLA(2)-IB, but not sPLA(2)-IA, demonstrated the presence of a sPLA(2)-R with saturation at 2.77 fM and a K(d) of 167 pM. sPLA(2)-R immunoreactivity was present at 185 kDa and localized to the membrane. Inactive sPLA(2)-IB activated p38 MAPK, and p38 MAPK inhibition attenuated elastase release. Active sPLA(2)-IA caused elastase release, but inactive type IA did not. sPLA(2)-IB stimulated elastase release independent of activity; inactive sPLA(2)-IIA partially stimulated PMNs. sPLA(2)-IB and sPLA(2)-IIA caused PMN adhesion. We conclude that PMNs contain a membrane M-type sPLA(2)-R that activates p38 MAPK.     

Exposure to static electric fields leads to changes in biogenic amine levels in the brains of Drosophila

Natural and anthropogenic static electric fields are commonly found in the environment and can have both beneficial and harmful effects on many animals. Here, we asked how the fruitfly responds to these fields and what the consequences of exposure are on the levels of biogenic amines in the brain. When given a choice in a Y-tube bioassay Drosophila avoided electric fields, and the greater the field strength the more likely Drosophila were to avoid it. By comparing wild-type flies, flies with wings surgically removed and vestigial winged flies we found that the presence of intact wings was necessary to produce avoidance behaviour. We also show that Coulomb forces produced by electric fields physically lift excised wings, with the smaller wings of males being raised by lower field strengths than larger female wings. An analysis of neurochemical changes in the brains showed that a suite of changes in biogenic amine levels occurs following chronic exposure. Taken together we conclude that physical movements of the wings are used by Drosophila in generating avoidance behaviour and are accompanied by changes in the levels of amines in the brain, which in turn impact on behaviour.     

Yin-yang ways of controlling gene expression are now in our hands.

Molecular biologists have long dreamed of switching genes on and off at will in any part of the body during embryonic development. Their dream is now coming true thanks to the transparency of the embryonic body of the zebrafish ( Danio rerio), which has made gene manipulation by photoillumination possible.     

Angiotensin II Triggers Expression of the Adrenal Gland Zona Glomerulosa-Specific 3β-Hydroxysteroid Dehydrogenase Isoenzyme through De Novo Protein Synthesis of the Orphan Nuclear Receptors NGFIB and NURR1

The 3β-hydroxysteroid dehydrogenase (3β-HSD) is an enzyme crucial for steroid synthesis. Two different 3β-HSD isoforms exist in humans. Classically, HSD3B2 was considered the principal isoform present in the adrenal. However, we recently showed that the alternative isoform, HSD3B1, is expressed specifically within the adrenal zona glomerulosa (ZG), where aldosterone is produced, raising the question of why this isozyme needs to be expressed in this cell type. Here we show that in both human and mouse, expression of the ZG isoform 3β-HSD is rapidly induced upon angiotensin II (AngII) stimulation. AngII is the key peptide hormone regulating the capacity of aldosterone synthesis. Using the human adrenocortical H295R cells as a model system, we show that the ZG isoform HSD3B1 differs from HSD3B2 in the ability to respond to AngII. Mechanistically, the induction of HSD3B1 involves de novo protein synthesis of the nuclear orphan receptors NGFIB and NURR1. The HSD3B1 promoter contains a functional NGFIB/NURR1-responsive element to which these proteins bind in response to AngII. Knockdown of these proteins and overexpression of a dominant negative NGFIB both reduce the AngII responsiveness of HSD3B1. Thus, the AngII-NGFIB/NURR1 pathway controls HSD3B1. Our work reveals HSD3B1 as a new regulatory target of AngII.     

Stable isotope-dilution liquid chromatography/tandem mass spectrometry method for determination of thyroxine in saliva

A liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) method for the determination of thyroxine (T₄) in human saliva has been developed and validated. The saliva was deproteinized with methanol, purified using a Strata-X? cartridge, and subjected to LC/ESI-MS/MS. Quantification was based on selected reaction monitoring, and [¹³C₆]-T₄ was used as the internal standard. This method allowed the reproducible (intra- and inter-assay relative standard deviations, <4.8%) and accurate (analytical recovery, 96.5–99.6%) quantification of the salivary T₄ using a 400 μl sample, and the limit of quantification was 25.0 pg/ml. A preliminary study using the developed method found that there is a diagnosable difference in the salivary T₄ concentration between the euthyroid subjects and the patients with Graves disease.     

The purification and complete amino acid sequence of the 9000-Mr Ca2+-binding protein from rat placenta. Identity with the vitamin D-dependent intestinal Ca2+-binding protein.

A 9000-Mr Ca2+-binding protein was isolated from rat placenta and purified to homogeneity by h.p.l.c. procedures. The complete amino acid sequence was established for the 78-residue placental protein. A sequence analysis of a minor component of the rat intestinal Ca2+-binding protein (residues 4-78) and a tryptic peptide (residues 55-74), both purified by h.p.l.c., showed both proteins to be identical. Thus this placental 9000-Mr Ca2+-binding protein is the same gene product as the intestinal Ca2+-binding protein whose synthesis is dependent on vitamin D.     

Molecular architecture of inner dynein arms in situ in Chlamydomonas reinhardtii flagella

The inner dynein arm regulates axonemal bending motion in eukaryotes. We used cryo-electron tomography to reconstruct the three-dimensional structure of inner dynein arms from Chlamydomonas reinhardtii. All the eight different heavy chains were identified in one 96-nm periodic repeat, as expected from previous biochemical studies. Based on mutants, we identified the positions of the AAA rings and the N-terminal tails of all the eight heavy chains. The dynein f dimer is located close to the surface of the A-microtubule, whereas the other six heavy chain rings are roughly colinear at a larger distance to form three dyads. Each dyad consists of two heavy chains and has a corresponding radial spoke or a similar feature. In each of the six heavy chains (dynein a, b, c, d, e, and g), the N-terminal tail extends from the distal side of the ring. To interact with the B-microtubule through stalks, the inner-arm dyneins must have either different handedness or, more probably, the opposite orientation of the AAA rings compared with the outer-arm dyneins.     

Cytological studies on rust fungi. (vi) fine structures of infection process of Kuehneola japonica (diet.) dietel

The behavior of rust fungi in their host plants has been elucidated by electron microscopy. However, most of the ultrastructural studies on rust fungi have focused on the uredial stage. In order to elucidate the features of the sporidial stage, we studied the fine structure of Kuehneola japonica, a short-cycle rust, in rose leaves. Infection pegs arising from appressoria penetrated the host walls. Papillae formed at the time of penetration against the outer epidermal cell walls. The papillae which had formed at the penetration sites grew extensively and partially surrounded the intracellular hyphae which were connected with the infection pegs. The intracellular hyphae in the epidermal cells developed further and entered adjacent parenchyma cells. Walls of parenchyma cells either invaginated or thin papillae formed at penetration sites and the invaginated walls or papillae surrounded the necks of the intracellular hyphae. Intracellular hyphae in both epidermal and parenchyma cells were not enveloped by the sheath before 20 days after inoculation. In specimens prepared 20 days after inoculation, some of the intracellular hyphae were enveloped by a sheath in both palisade and spongy parenchyma cells. The sheathed hyphae resembled haustoria of other rust fungi which had been described previously. Teliospore initials were formed in mycelial masses in intercellular spaces between the epidermal cells and palisade parenchyma cells 20 days after inoculation. Uninucleate teliospores developed from teliospore initials 30 days after inoculation.Contribution No. 32.     

Expression and relationship of male reproductive ADAMs in mouse

A number of a disintegrin and metalloprotease (ADAM) family members are expressed in mammalian male reproductive organs such as testis and epididymis. These reproductive ADAMs are divided phylogenically into three major groups: ADAMs 1, 4, 6, 20, 21, 24, 25, 26, 29, 30, and 34 (the first group); ADAMs 2, 3, 5, 27, and 32 (the second group); and ADAMs 7 and 28 (the third group). Previous mouse knockout studies indicate that ADAM1, ADAM2, and ADAM3 have intricate expressional relationships, playing critical roles in fertilization. In the present study, we analyzed processing, biochemical characteristics, localization, and expressional relationship of the previously-unexplored, second-group ADAMs (ADAM5, ADAM27, and ADAM32). We found that all of the three ADAMs are made as precursors in the testis and processed during epididymal maturation, and that ADAM5 and ADAM32, but not ADAM27, are located on the sperm surface. Using sperm from Adam2(-/-) and Adam3(-/-) mice, we found that, among the three ADAMs, the level of ADAM5 is modestly and severely reduced in Adam3 and Adam2 knockout sperm, respectively. Further, we analyzed ADAM7, an epididymis-derived sperm surface ADAM from the separate phylogenetic group, in the knockout sperm. We found that the level of ADAM7 is also significantly reduced in both Adam2 and Adam3-null sperm. Taken together, our results suggest a novel expressional relationship of ADAM5 and ADAM7 with ADAM2 and ADAM3, which play critical roles in fertilization.