Interstrand crosslink-induced radials form between non-homologous chromosomes, but are absent in sex chromosomes

Fanconi anemia (FA) and cells lacking functional BRCA1 and BRCA2 proteins are hypersensitive to interstrand crosslinking (ICL) agents and show increased numbers of chromosomal breaks and radials. Although radial formation has been used to diagnose FA for more than 30 years, there has been little analysis of these characteristic formations. In this study, radials were analyzed from FA-A and FA-G fibroblasts as well as normal and retrovirally-corrected FA-A fibroblasts treated with high doses of ICLs. Radials were found to only involve non-homologous chromosome interactions and to be distributed nearly randomly along the length of chromosomes. Sites on chromosomes that did show increased frequency of radial involvement did not correlate with known fragile sites or pericentric regions. Hybrid radials were observed between mouse and human chromosomes in human-mouse hybrid cells produced by microcell-mediated chromosome transfer of mouse chromosomes into human FA-A fibroblasts. Both X and Y chromosomes were notably not involved in radials. These observations suggest that ICL repair may involve short stretches of homology, resulting in aberrant radial formation in the absence of FA proteins.     

Water channel aquaporin-2 directly binds to actin

Water channel aquaporin-2 (AQP2) strictly regulates body water homeostasis in mammals. Trafficking of AQP2 to the apical membrane is critical to the reabsorption of water in renal collecting ducts. Controlled apical positioning of AQP2 suggests the interaction of AQP2 with other proteins. To isolate AQP2-binding proteins, immunoaffinity chromatography of extracts from rat kidney papilla was performed using a column covalently coupled with anti-AQP2 antibody. Using this method 42-kDa protein was purified and subsequently identified as beta- and gamma-isoforms of actin by two-dimensional gel analysis and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. AQP2 was indeed coimmunoprecipitated with actin from cell lysates of rat kidney papilla. In addition, surface plasmon resonance analyses showed that the C-terminal fragment of AQP2 strongly bound to actin and the K(D) value was 3.18x10(-8)M. In this experiment we have elucidated the direct binding of channel protein AQP2 to cytoskeletal protein actin, providing a novel mechanism for trafficking of not only AQP2 but also recycling channel proteins.     

Production of N-lauroylated G protein alpha-subunit in Sf9 insect cells: the type of N-acyl group of Galpha influences G protein-mediated signal transduction

The alpha-subunit of rod photoreceptor G protein transducin (G(t1)alpha) is heterogeneously modified at the N-terminus by a mixture of acyl groups, laurate (C12:0), myristate (C14:0), and two unsaturated fatty acids (C14:1 and C14:2). Although the N-fatty acylation of G(t1)alpha plays important roles in protein-protein and protein-membrane interactions in light signaling, the biological significance of the heterogeneous acylation remains unclear due to the difficulty in isolating each G(t1)alpha isoform from the retinal rod cells. Here we found that G(t1)alpha/G(i1)alpha chimera (G(t/i)alpha) expressed in Sf9 cells is also heterogeneously modified by myristate (approximately 90%) and laurate (approximately 10%), raising the possibility that the N-acyl group of recombinant G(t/i)alpha may be manipulated by modifying culture media. In fact, addition of myristic acid to the medium decreased the relative content of lauroylated G(t/i)alpha to an undetectable level, whereas exogenously added lauric acid significantly increased the relative content of lauroylated G(t/i)alpha in a concentration-dependent manner. By culturing the G(t/i)alpha-virus infected Sf9 cells with fatty acids, we obtained four different preparations of N-acylated G(t/i)alpha, in which the relative abundance of lauroylated isoform was 0%, 20%, 33% and approximately 70%, respectively. Functional analysis of these proteins showed that an increase in the relative content of the lauroylated isoform remarkably slowed down the steady-state GTP hydrolysis rate of G(t/i)alpha; the steady-state GTPase activity of the lauroylated isoform was estimated to be one order of magnitude lower than that of the myristoylated isoform. These results suggest that the retinal G(t1)alpha is composed of isoforms having functionally heterogeneous signaling properties.     

Effects of partial agonists and Mg2+ ions on the interaction of M2 muscarinic acetylcholine receptor and G protein Galpha i1 subunit in the M2-Galpha i1 fusion protein

We have expressed a M(2)-Galpha(i1) fusion protein in insect cells, in which the G protein alpha(i1) subunit was fused with a mutant of the muscarinic receptor M(2) subtype without glycosylation sites and the central part of the third intracellular loop. The M(2)-Galpha(i1) fusion protein showed GTP-sensitive, high-affinity agonist binding. Displacement curves by GDP of [(35)S]GTPgammaS binding shifted to the right in the presence of muscarinic agonists. The extent of the shift was greater for full agonists (120-150 fold) than for partial agonists (25-35 fold), and virtually no shift was observed for antagonists. The affinity for GDP decreased with increasing MgCl(2) concentration in the presence of an agonist but was not affected by MgCl(2) in the presence of an antagonist. These results indicate that the apparent affinity for GDP of the M(2)-Galpha(i1) fusion protein bound to a ligand represents the efficacy of the given ligand, and that Mg(2+) is required for the agonist-bound M(2) to interact with Galpha(i1), reducing its affinity for GDP. We propose that the agonist-M(2)-Galpha(i1) complex represents the transition state for the GDP-GTP exchange reaction catalyzed by agonist-bound receptors, and that the complex has different affinities for GDP depending on the species of the ligand bound to M(2) receptors.     

Light-harvesting properties of zinc complex of chlorophyll-<Emphasis Type="Italic">a</Emphasis> from <Emphasis Type="Italic">spirulina</Emphasis> in surfactant micellar media

Zn chlorophyll-a was prepareted from Mg chlorophyll-a from spirulina and the optical properties of the ground state and the photoexited state of Zn chlorophyll-a in aqueous surfactant micellar media were studied using UV-vis absorption, fluorescence emission spectra, electrochemical and fluorescence lifetime measurements. In comparison of the UV-vis absorption and fluorescence emission spectra of Zn chlorophyll-a and Mg chlorophyll-a, the blue-shift in the absorption bands and emission peak of Zn chlorophyll-a was observed. The energies of the first excited singlet state of Zn chlorophyll-a was 1.87eV. The first oxidation and reduction potentials of the photoexcited singlet state of Zn chlorophyll-a were −0.67 and 0.60V, respectively. Fluorescence lifetime of Zn chlorophyll-a was 9.0 ns in CTAB micellar solution. The fluorescence lifetime of Zn chlorophyll-a is shorter than that of Mg chlorophyll-a (9.8 ns). The photositability of Zn chlorophyll-a was superior to that of Mg chlorophyll-a in various pH conditions. Published online December 2004     

Fatal Baylisascaris larva migrans in a colony of Japanese macaques kept by a safari-style zoo in Japan

A colony of Japanese macaques (Macaca fuscata fuscata) kept by a safari-style zoo in Japan experienced 9 sporadic cases of fatal neurological diseases, such as epilepsy and posterior paralysis, during the 12 yr from 1989 to 2001. This macaque colony consisted of approximately 30 animals, on average, during this period, and the macaques shared their living space with II American black bears (Ursus americanus) harboring zoonotic roundworms (Baylisascaris transfuga). Close to this enclosure, a cote for 2-3 raccoons (Procyon lotor) was placed, and raw sewage from this cote ran into a shallow drain in the area for macaques and bears. However, fecal examinations in recent years did not detect the infection of raccoons with zoonotic roundworms (Baylisascaris procyonis). Postmortem histological examination of the latest 2 ill macaques detected multifocal malacia in the brain; 2 ascarid larvae of 60 microm maximum width were encapsulated in the cerebrum and lungs of 1 of the animals. To determine the causative ascarid species of the fatal larva migrans, we analyzed 2 additional encapsulated Baylisascaris larvae collected from formalin-fixed lungs by morphological and molecular approaches. This sporadic outbreak is the second record of Baylisascaris larva migrans in animals in Japan.     

Role of thymic output in regulating CD8 T-cell homeostasis during acute and chronic viral infection

Although it is well documented that CD8 T cells play a critical role in controlling chronic viral infections, the mechanisms underlying the regulation of CD8 T-cell responses are not well understood. Using the mouse model of an acute and chronic lymphocytic choriomeningitis virus (LCMV) infection, we have examined the relative importance of peripheral T cells and thymic emigrants in the elicitation and maintenance of CD8 T-cell responses. Virus-specific CD8 T-cell responses were compared between mice that were either sham thymectomized or thymectomized (Thx) at approximately 6 weeks of age. In an acute LCMV infection, thymic deficiency did not affect either the primary expansion of CD8 T cells or the proliferative renewal and maintenance of virus-specific lymphoid and nonlymphoid memory CD8 T cells. Following a chronic LCMV infection, in Thx mice, although the initial expansion of CD8 T cells was normal, the contraction phase of the CD8 T-cell response was exaggerated, which led to a transient but striking CD8 T-cell deficit on day 30 postinfection. However, the virus-specific CD8 T-cell response in Thx mice rebounded quickly and was maintained at normal levels thereafter, which indicated that the peripheral T-cell repertoire is quite robust and capable of sustaining an effective CD8 T-cell response in the absence of thymic output during a chronic LCMV infection. Taken together, these findings should further our understanding of the regulation of CD8 T-cell homeostasis in acute and chronic viral infections and might have implications in the development of immunotherapy.     

Seven skeletal muscles rich in slow muscle fibers may function to sustain neutral position in the rodent hindlimb

Skeletal muscles consist of slow-twitch and fast-twitch muscle fibers, which have distinct physiological and biochemical properties. The muscle fiber composition determines the contractile velocity and fatigability of a particular skeletal muscle. We analyzed the systemic distribution of slow muscle fibers in all rodent skeletal muscles by myosin ATPase staining and found that only seven hindlimb skeletal muscles were extremely rich in slow muscle fibers. These included the mouse piriformis (56.5%), gluteus minimus (35.7%), vastus intermedius (24.7%), quadratus femoris (69.9%), adductor brevis (44.3%), gracilis (24.6%), and soleus muscles (35.1%). In mice, the relative proportion of slow muscle fibers did not exceed 15% in skeletal muscles in other regions. The distribution of slow muscle fibers was well conserved in rats and rabbits. The soleus muscle is an important antigravity muscle in both rodents and humans; therefore, these skeletal muscles rich in slow muscle fibers might play an important role in sustaining neutral alignment of the lower extremity.     

MAPKs (ERK1/2, p38) and AKT can be phosphorylated by shear stress independently of platelet endothelial cell adhesion molecule-1 (CD31) in vascular endothelial cells

PECAM-1 (CD31) is a member of the Ig superfamily of cell adhesion molecules and is expressed on endothelial cells (EC) as several circulating blood elements including platelets, polymorphonuclear leukocytes, monocytes, and lymphocytes. PECAM-1 tyrosine phosphorylation has been observed following mechanical stimulation of EC but its role in mechanosensing is still incompletely understood. The aim of this study was to investigate the involvement of PECAM-1 in signaling cascades in response to fluid shear stress (SS) in vascular ECs. PECAM-1-deficient (KO) and PECAM-reconstituted murine microvascular ECs, 50 and 100% confluent bovine aortic EC (BAEC), and human umbilical vein EC (HUVEC) transfected with antisense PECAM-1 oligonucleotides were exposed to oscillatory SS (14 dynes/cm2) for 0, 5, 10, 30 or 60 min. The tyrosine phosphorylation level of PECAM-1 immunoprecipitated from SS-stimulated PECAM-reconstituted, but not PECAM-1-KO, murine ECs increased. Although PECAM-1 was phosphorylated in 100% confluent BAEC and HUVEC, its phosphorylation level in 50% confluent BAECs or HUVEC was not detected by SS. Likewise PECAM-1 phosphorylation was robust in the wild type and scrambled-transfected HUVEC but not in the PECAM-1 antisense-HUVEC. ERK(1/2), p38 MAPK, and AKT were activated by SS in all cell types tested, including the PECAM-1-KO murine ECs, 50% confluent BAECs, and HUVEC transfected with antisense PECAM-1. This suggests that PECAM-1 may not function as a major mechanoreceptor for activation of MAPK and AKT in ECs and that there are likely to be other mechanoreceptors in ECs functioning to detect shear stress and trigger intercellular signals.