Possible testosterone redundancy for 5α-dihydrotestosterone in the masculinization of mouse external genitalia

The development of embryonic external genitalia (eExG) into characteristic male structures, such as urethra and penile erectile tissues, depends on 5α-dihydrotestosterone (DHT). Although the corpus cavernosum (CC) is well known as essential for erectile function in adults, its developmental process and its dependency on DHT have been unknown. To reveal the dimorphic formation of the murine CC from the embryonic stage, we first analyzed the production of the protein vascular endothelial growth factor receptor-2 (FLK1) via its expression (hereinafter referred as “expression of FLK1”) and the expression of alpha-smooth muscle actin (ACTA2) and collagen type 1 (COL1A1) in developing external genitalia. The 5-α reductase type 2 encoded by the SRD5A2 gene has been suggested to be a crucial enzyme for male sexual differentiation, as it converts testosterone (T) into DHT in the local urogenital organs. In fact, SRD5A2 mutation results in decreased synthesis of DHT, which leads to various degrees of masculinized human external genitalia (ExG). We further investigated the expression profile of SRD5A2 during the formation of the murine CC. We observed that SRD5A2 was expressed in smooth muscle of the CC. To determine the role of SRD5A2 in CC formation, we analyzed the formation of erectile tissue in the male Srd5a2 KO mice and measured the levels of androgens in the ExG by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Intriguingly, there were no obvious defects in the CCs of male Srd5a2 KO mice, possibly due to increased T levels. The current study suggests possible redundant functions of androgens in CC development.     

Annual changes in serum levels of two choriogenins and vitellogenin in masu salmon, Oncorhynchus masou

Annual changes in serum levels of two chorion precursors, choriogenin H (Chg H) and choriogenin L (Chg L), vitellogenin (Vg) and estradiol-17beta (E2) were quantified in masu salmon, Oncorhynchus masou, using specific immunoassays. Serum Chg levels were higher than Vg during the previtellogenic growth phase when circulating E2 levels were low ( approximately 0.1 ng/mL), suggesting higher sensitivity of Chg to E2. When oocyte growth shifted to the vitellogenic phase, Vg levels increased and became the most abundant in serum coincident with elevations of E2 and GSI. Chg H, Chg L and Vg peaked 1 month prior to ovulation at 0.61+/-0.08, 0.98+/-0.18 and 10.93+/-3.24 mg/mL, respectively. These results suggest that chorion formation by Chgs occurs prior to vitellogenesis and that the sensitivity of Chgs to low circulating E2 is closely related to the sequential events of oocyte growth.     

Secondary Cell Wall Formation: Changes in Cell Wall Constituents during the Differentiation of Isolated Mesophyll Cells of Zinnia elegans to Tracheary Elements

Changes in the cell walls and their sugar composition duringthe formation of tracheary elements (TE) were analyzed usinga culture of single cells isolated from the mesophyll of Zinniaelegans. By using Calcofluor White the first differentiatingcells were observed 36 to 38 h after the start of culture. Thisis 8 to 10 hours before differentiating cells can be observedwithout staining, and about 14 to 16 hours before the beginningof lignification of differentiating cells. In correlation withthe appearance of differentiating cells, the following changeswere observed: (1) a significant increase in the total carbohydratein the 5% KOH-soluble, the 24% KOH-soluble and insoluble cellulosicfractions; (2) a decrease in the relative amount of uronic acidsin the EDTA-soluble fraction which corresponded to increasesin the KOH-soluble fractions and in the insoluble fraction;(3) an enormous increase in the absolute and relative amountof xylose in the hemicellulosic fractions and to some extentalso in the cellulosic fraction. Methylation analysis indicatedthat the high amount of xylose reflects the synthesis of a xylan-typepolysaccharide which is deposited simultaneously with celluloseprior to the lignification of the wall. (Received August 5, 1987; Accepted December 9, 1987)     

A mitochondria-selective near-infrared-emitting fluorescent dye for cellular imaging studies

This communication details the synthesis, evaluation of photophysical properties, and cellular imaging studies of cyanine chromophore based fluorescent dye 1 as a selective imaging agent for mitochondria.     

Synthetic sialylphosphatidylethanolamine derivatives bind to human influenza A viruses and inhibit viral infection

We synthesized the sialylphosphatidylethanolamine (sialyl PE) derivatives Neu5Ac-PE, (Neu5Ac)2-PE, Neu5Ac-PE (amide) and Neu5Ac-PE (methyl). We examined the anti-viral effects of the derivatives on human influenza A virus infection by ELISA/virus-binding, hemagglutination inhibition, hemolysis inhibition and neutralization assays. The sialyl PE derivatives that we examined bound to A/Aichi/2/68, A/Singapore/1/57 and A/Memphis/1/71 strains of H3N2 subtype, but not to A/PR/8/34 strain of H1N1 subtype. The derivatives inhibited viral hemagglutination and hemolysis of human erythrocytes with A/Aichi/2/68 and A/Singapore/1/57 (H3N2), but not with A/PR/8/34 (H1N1). The inhibitory activity of the (Neu5Ac)2-PE derivative was the strongest of all sialyl PE derivatives (IC50, 35 M to 40 M). Sialyl PE derivatives also inhibited the infection of A/Aichi/2/68 in MDCK cells. Complete inhibition was observed at a concentration between 0.3 to 1.3 mM. IC50 of (Neu5Ac)2-PE was 15 M in A/Aichi/2/68 strain. Taken together, the synthetic sialyl PE derivatives may be effective reagents against infection of some types of influenza A viruses.     

Separation and characterization of the isoenzymes of wall-bound peroxidase from cultured Zinnia cells during tracheary element differentiation

Cell wall-bound peroxidase (EC 1.11.1.7) isoenzymes (P1-P5) from cells of Zinnia elegans L. that were differentiating into tracheary elements were separated and characterized to obtain information about the relationships between these isoenzymes and the biosynthesis of lignin. Fractionation of Zinnia cells by centrifugation in solutions of Percoll revealed that P1, P2, and P5 were present in differentiated tracheary elements. These peroxidase isoenzymes were separated by several column-chromatographic steps. During hydrophobic chromatography on Phenyl Superose, P5 activity was separated into activities P5A and P5B. Enzymatically pure preparations of P1, P3, P5A, and P5B were finally obtained and used for the characterization of each isoenzyme. The optimum pH was 5.5–6.0 for P1, 5.0–7.5 for P3, 5.0 for P5A, and 4.0 for P5B. Each of the isoenzymes oxidized coniferyl alcohol efficiently, whereas p-coumaryl alcohol and sinapyl alcohol were poor substrates for all the isoenzymes. An absolute requirement for Ca²⁺ ions was demonstrated for P3. Based on these results, possible roles of peroxidase isoenzymes in the formation of lignin during the differentiation of tracheary elements are discussed.Abbreviations DAB diaminobenzidine - GTA equal proportions of 3,3-dimethylglutaric acid, tris(hydroxymethyl)aminomethane, and 2-amino-2-methyl-1,3-propanediol - TE tracheary element The authors are very grateful to Professor M. Tanahashi of Gifu University for providing hydroxycinnamyl alcohols. This work was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan to H.F.     

The Protein Scaffold NHERF-1 Controls the Amplitude and Duration of Localized Protein Kinase D Activity

Protein kinase D (PKD) transduces an abundance of signals downstream of diacylglycerol production. The mammalian PKD family consists of three isoforms, PKD1, PKD2, and PKD3; of these PKD1 and PKD2 contain PDZ-binding motifs at their carboxyl termini. Here we show that membrane-localized NHERF scaffold proteins provide a nexus for tightly controlled PKD signaling via a PDZ domain interaction. Using a proteomic array containing 96 purified PDZ domains, we have identified the first PDZ domain of NHERF-1 as an interaction partner for the PDZ-binding motifs of both PKD1 and PKD2. A fluorescence resonance energy transfer-based translocation assay reveals a transient association of PKD1 and PKD2 with NHERF-1 in live cells that is triggered by phorbol ester stimulation and, importantly, differs strikingly from the sustained translocation to plasma membrane. Targeting a fluorescence resonance energy transfer-based kinase activity reporter for PKD to NHERF scaffolds reveals a unique signature of PKD activation at the scaffold that is distinct from that of general cytosolic or plasma membrane activity. Specifically, agonist-evoked activation of PKD at the scaffold is rapid and sustained but blunted in magnitude when compared with cytosolic PKD. Thus, live cell imaging of PKD activity demonstrates ultrasensitive control of kinase signaling at the scaffold compared with bulk activity in the cytosol or at the plasma membrane.Protein kinase D (PKD)² plays a role in numerous processes including cell proliferation, cell survival, immune cell signaling, gene expression, vesicle trafficking, and neuronal development (1). The PKD family consists of three members belonging to the Ca²⁺/calmodulin-dependent kinase group of serine/threonine protein kinases. Each isoform contains a conserved catalytic core and an amino-terminal regulatory moiety. This regulatory region contains two cysteine-rich (C1) domains and a pleckstrin homology domain that autoinhibits the kinase (2). The C1 domains are membrane-targeting modules that bind diacylglycerol (DAG) and its functional analogues, phorbol esters, thus recruiting PKD to membranes (3). The PKD1 and PKD2 isoforms additionally contain PDZ-binding motifs at their carboxyl termini that can target the kinases to distinct subcellular scaffolds through interactions with PDZ domain-containing proteins (4).PKD transduces signals downstream of the second messenger DAG. In addition to membrane recruitment by DAG, activation of PKD requires phosphorylation by novel protein kinase C (PKC) family members at two sites within its catalytic core (5, 6). The novel PKCs themselves contain C1 domains and are allosterically activated by DAG-mediated membrane binding; thus, DAG production leads to PKD activation through coincident activation of the novel PKCs and localization of PKD near its upstream kinases. Hence, activation of phospholipase C (PLC)-coupled receptors (such as certain G protein-coupled receptors (GPCRs) or receptor tyrosine kinases) results in the production of second messengers including DAG, and this leads to recruitment and activation of the novel PKCs and thus also PKD.PDZ (PSD-95, Discs large, ZO-1) domains are compact, globular structures of ∼90 residues, occurring in one or multiple copies within a protein, that mediate protein-protein interactions (7). These interactions occur via binding to other PDZ domains or, more commonly, by recognition of short amino acid motifs in the carboxyl termini of target proteins commonly terminating in a hydrophobic residue (8). In the case of PKD1 and PKD2, the last four amino acids are VSIL and ISVL, respectively. Here we identify Na⁺/H⁺ exchanger regulatory factor 1 (NHERF-1) as a PDZ domain-containing protein that interacts with the PDZ-binding motif of both PKD1 and PKD2.NHERF-1 was originally cloned as a critical protein component for the inhibition of Na⁺/H⁺ exchanger 3 by protein kinase A (9). NHERF-1 is 52% identical to NHERF-2, a family member with which it shares the conserved domain structure of two PDZ domains followed by an ezrin-radixin-moesin (ERM)-binding region (10). Parallel studies demonstrating its ability to strongly interact with ezrin independently identified NHERF-1 as ERM-binding phosphoprotein 50 (11). Via this ERM-binding region, NHERF-1 and NHERF-2 are predominantly localized near the actin cytoskeleton, thus poising them near the plasma membrane where they function as scaffolds. Since these original cloning reports, numerous studies have identified over 30 binding partners of these scaffold proteins including GPCRs, tyrosine kinase receptors, other adaptor proteins, signaling enzymes, and ion channels (12, 13).Here we identify PKD1 and PKD2 as NHERF-1-interacting proteins. Using a fluorescence resonance energy transfer (FRET)-based assay to assess molecular proximity, both PKD1 and PKD2 are shown to transiently associate with NHERF-1 following PKD activation. Furthermore, through use of genetically encoded reporters for PKD activity, we show a unique signature of PKD activation at the NHERF scaffold. Specifically, signaling is more tightly regulated at the scaffold than in the cytosol or bulk plasma membrane. Phosphatase activity is higher at NHERF than at the plasma membrane, resulting in a more rapidly reversible PKD response at the scaffold, and following an agonist-evoked response, PKD signaling is prolonged compared with the length of response in the cytosol. Our data identify NHERF-1 as a novel nexus of PKD signaling and raise the possibility that PKD may act as a novel regulator of proteins at the NHERF scaffold.     

Purification and identification of a second form of vitellogenin from ascites of medaka (Oryzias latipes) treated with estrogen

Estrogen treatment of medaka leads to accumulation of ascites, in which vitellogenin (Vg) and choriogenins (precursors to vitelline envelope) are abundant. Besides those female-specific proteins, we detected a new component in ascites that cross-reacts with antiserum against egg yolk proteins. We tentatively named it egg yolk-related protein (YRP). YRP was purified from ascites by hydroxylapatite chromatography followed by gel filtration. Purified YRP had a molecular mass of 460 kDa in intact state while 570 kDa for Vg. The molecular weight of purified YRP on SDS-PAGE under both reducing and nonreducing conditions was 130 kDa. YRP was confirmed to be a lipoglycophosphoprotein by staining with Sudan black, periodic acid-Schiff (PAS) and methyl green. Amino acid composition of YRP resembled that of Vg except for a relatively low content of serine. A specific antiserum against YRP was raised in a rabbit. Antiserum against YRP specifically immunostained its antigen but not Vg or choriogenins. YRP was detected as a female-specific protein in serum of breeding medaka. The antiserum also cross-reacted with a band at 29 kDa in egg extracts, which is not immunoreactive to antiserum against Vg. These data show that YRP is a precursor to some egg yolk proteins with differing antigenicity from Vg (Hamazaki et al. '87). We thus conclude that YRP is a second form of medaka Vg and rename YRP as Vg 2 while formerly reported Vg as Vg 1.