Vibration receptive sensilla on the wing margins of the silkworm moth Bombyx mori

Bristles along the wing margins (wm-bristles) of the silkworm moth, Bombyx mori, were studied morphologically and electrophysiologically. The male moth has ca. 50 wm-bristles on each forewing and hindwing. Scanning electron microscopy revealed that these wm-bristles are typical mechanosensilla. Leuco-methylene blue staining demonstrated that each wm-bristle has a single receptor neuron, which is also characteristic of the mechanosensillum. The receptor neuron responded to vibrating air currents but did not respond to a constant air current. The wm-bristles showed clear directional sensitivity to vibrating air currents. The wm-bristles were classified into two types, type I and type II, by their response patterns to sinusoidal movements of the bristle. The neuron in type I discharged bursting spikes immediately following stimulation onset and also discharged a single spike for each sinusoidal cycle for frequencies less than ca. 60 Hz. The neuron in type II only responded to vibrations over 40 Hz and, specifically at 75 Hz, discharged a single spike for each sinusoidal cycle throughout the stimulation period. These results suggest that the two types of wm-bristles are highly tuned in different ways to detect vibrations due to the wing beat. The roles of the wm-bristles in the wing beat are discussed.     

The Cytoplasmic Domain of Human Immunodeficiency Virus Type 1 Transmembrane Protein gp41 Harbors Lipid Raft Association Determinants

The molecular basis for localization of the human immunodeficiency virus type 1 envelope glycoprotein (Env) in detergent-resistant membranes (DRMs), also called lipid rafts, still remains unclear. The C-terminal cytoplasmic tail of gp41 contains three membrane-interacting, amphipathic α-helical sequences, termed lentivirus lytic peptide 2 (LLP-2), LLP-3, and LLP-1, in that order. Here we identify determinants in the cytoplasmic tail which are crucial for Env''s association with Triton X-100-resistant rafts. Truncations of LLP-1 greatly reduced Env localization in lipid rafts, and the property of Gag-independent gp41 localization in rafts was conserved among different strains. Analyses of mutants containing single deletions or substitutions in LLP-1 showed that the α-helical structure of the LLP-1 hydrophobic face has a more-critical role in Env-raft associations than that of the hydrophilic face. With the exception of a Pro substitution for Val-833, all Pro substitution and charge-inverting mutants showed wild-type virus-like one-cycle viral infectivity, replication kinetics, and Env incorporation into the virus. The intracellular localization and cell surface expression of mutants not localized in lipid rafts, such as the TM844, TM813, 829P, and 843P mutants, were apparently normal compared to those of wild-type Env. Cytoplasmic subdomain targeting analyses revealed that the sequence spanning LLP-3 and LLP-1 could target a cytoplasmic reporter protein to DRMs. Mutations of LLP-1 that affected Env association with lipid rafts also disrupted the DRM-targeting ability of the LLP-3/LLP-1 sequence. Our results clearly demonstrate that LLP motifs located in the C-terminal cytoplasmic tail of gp41 harbor Triton X-100-resistant raft association determinants.Lentiviruses, including human immunodeficiency virus type 1 (HIV-1), are unusual in possessing a long cytoplasmic domain (∼150 amino acids) in their envelope (Env) transmembrane (TM) glycoprotein compared to those of other retroviruses (20 to 50 amino acids). The cytoplasmic domain of HIV-1 TM protein gp41, which encompasses residues 706 to 856, has multiple functions during the virus life cycle, including viral replication, infectivity, transmission, and cytopathogenicity. Truncations of the HIV-1 cytoplasmic domains may modulate cell-cell fusion properties of the Env protein, presumably due to alterations in the levels of cell surface Env expression and conformation of the Env ectodomain (23, 81). The cytoplasmic domain is characterized by the presence of three structurally conserved, amphipathic α-helical segments, located at residues 828 to 856, 770 to 795, and 786 to 813 and referred to as lentivirus lytic peptide 1 (LLP-1), LLP-2, and LLP-3, respectively, at its C terminus (Fig. ​(Fig.1A).1A). The LLP-1 and LLP-2 sequences were shown to be inserted into viral membranes by a photoinduced chemical reaction (73). These LLP motifs have been implicated in a variety of functions, such as cell surface expression (12), Env fusogenicity (30), and Env incorporation into a virus (47, 56), as well as Env protein stability (33) and multimerization (34).Open in a separate windowFIG. 1.(A) Schematic representation of the gp41 cytoplasmic domain and truncation mutants examined in this study. The cytoplasmic tail of gp41 contains a tyrosine-based endocytic YSPL signal located at residue 712, a hydrophilic region, a diaromatic YW motif, and three amphipathic α-helices, termed LLP-2, LLP-3, and LLP-1, at its C terminus. The amino acid sequence from residues 806 to 856 of the WT HXB2 Env is presented in single amino acid code, and the C-terminal dileucine motif is underlined in the sequence. Truncation mutants (TMs) generating stop codons immediately downstream of the indicated amino acids and their respective sequences are also shown. (B) pHXB2R3-based mutant proviruses used in this study. All mutants were generated by a PCR overlap cloning strategy, and the mutation sites are indicated. A dash or dot indicates that the residue in that position of the mutant provirus sequence is identical to or absent from that of the WT provirus sequence, respectively. The substituted amino acids in the mutant proviruses are also indicated.Gag and Env carry specific intracellular localization signals governing the site(s) of virus assembly/budding and release into the extracellular milieu. Env trafficking to the plasma membrane is regulated by the conserved C-terminal dileucine motif and the endocytic, membrane-proximal, tyrosine-based GY₇₁₂SPL signal in the cytoplasmic tail of gp41 (Fig. ​(Fig.1A)1A) and by their respective interactions with the clathrin adaptor proteins, AP1 and AP2 (4, 9, 21, 49, 65, 77). A diaromatic motif, Y₈₀₂W₈₀₃, was shown to bind to TIP47, a protein required for the retrograde transport of mannose-6-phosphate receptors from late endosomes to the trans-Golgi network, and this interaction was involved in the retrograde transport of Env to the trans-Golgi network (8). Alterations of these intracellular localization signals may affect viral infectivity, Env assembly into the virus, and viral replication (8, 20). Likewise, Gag also contains important sequences required for its trafficking to and assembly at the plasma membrane. The matrix (MA) protein, p17, contains a myristoyl group and a cluster of basic amino acids, while p6 contains a late domain which interacts with the components of the endosomal sorting complex required for transport (ESCRT) pathway to mediate Gag trafficking to the virion assembly/budding site (for reviews, see references 25, 45, 57, and 59). It is well documented that the specific interaction between the cytoplasmic domain of gp41 and the trimeric MA protein in infected cells facilitates recruitment of the Env into virus assembly/budding sites on target membranes (for reviews, see references 18, 24, and 46). TIP47 was demonstrated to act as an adaptor to bridge the gp41 cytoplasmic domain and Gag, which allows the physical encounter between Gag and Env, resulting in efficient Env incorporation into the virus during the viral assembly/budding process (39).Lipid rafts, also called detergent-resistant membranes (DRMs), are highly specialized membrane microdomains present in both the plasma and endosomal membranes of eukaryotic cells. These dynamic microdomains are characterized by their detergent insolubility, light density on a sucrose gradient, and enrichment of cholesterol, glycosphingolipids, and glycosylphosphatidylinositol (GPI)-linked proteins that are anchored in the membrane by their attached GPI moieties (1). HIV-1 utilizes lipid rafts to efficiently enter host cells (40, 74, 80) and selectively assembles and buds from lipid rafts on the surfaces of infected cells (27, 36, 48, 50, 54). Also, the HIV-1 Env protein was detected in lipid raft membranes (48, 54, 64). Lipid rafts are thought to facilitate Env-Gag interactions, to concentrate viral Env glycoproteins, and to promote multimerization of intracellular viral components (for a review, see reference 51). However, what governs Env transport to and localization in lipid rafts is a long-standing question.Although the mechanisms by which proteins associate with lipid rafts are not fully understood, determinants for targeting of signal proteins to DRMs have been identified. These include a GPI anchor (2, 61) and an N-terminal Met-Gly-Cys in which Gly is myristylated and Cys is palmitoylated (43, 71). The latter includes certain dually acylated heterotrimeric guanine nucleotide-binding protein (G protein) α subunits (44). In addition, acylation by palmitoylation also serves as a signal to target signaling molecules to lipid rafts (for reviews, see references 11 and 60). Some Env proteins of membrane-enveloped viruses are known to be associated with lipid rafts (35, 41, 54, 69, 79), and acylation of viral Env proteins, in particular, palmitoylation, is important for targeting these Env proteins to lipid rafts (for reviews, see references 58 and 70).It is generally believed that the association of HIV-1 Env with lipid rafts requires a palmitoylation signal(s) located in the cytoplasmic tail of gp41 (6, 64). Nevertheless, the two cytoplasmic palmitoylated Cys residues in the HXB2 strain Env protein are not conserved among HIV-1 isolates, and some isolates do not even contain cysteine residues in their cytoplasmic tail (32). In accordance with this notion, we previously demonstrated that the two cytoplasmic palmitoylated Cys residues in T-cell (T)- and macrophage (M)-tropic Env proteins do not play an obvious role in the virus life cycle, including Env''s association with lipid rafts (13), suggesting that other factors may substitute for cytoplasmic palmitoylation to promote Env localization in lipid rafts. Clapham''s group showed that mutations in MA or the cytoplasmic tail that prevent Env from incorporating into the virus and impair virus infectivity also interfere with Env''s association with lipid rafts (7), indicating that the Gag-Env interaction drives efficient Env association with lipid rafts, which in turn modulates Env budding and assembly onto viral particles. In contrast to their findings, we previously also noted that the Env protein of the HXB2 strain expressed without Gag is still located in lipid rafts (13), providing compelling evidence for the proposal that the Env per se contains sufficient information for its sequestration into lipid rafts.To further understand the nature of Env''s association with lipid rafts, in the present study we show that sequestering Env in Triton X-100-resistant lipid rafts is an intrinsic property of Env and is independent of Gag-Env interactions. Additionally, the LLP motifs, in particular the α-helical structure of the hydrophobic face of LLP-1, play a crucial role in Env''s localization in lipid rafts. Except for the 833P mutant of Env, which is unstable and degrades (33), all Pro-substituted mutants not located in lipid rafts exhibited wild-type (WT)-like phenotypes of intracellular localization, cell surface expression, incorporation into virions, and viral replication capacity. Importantly, the α-helix of the hydrophobic face of LLP-1 is also critical for the raft-targeting ability of the LLP-3/LLP-1 sequence. Our study depicts, for the first time, the critical role of the α-helix of the gp41 cytoplasmic domain in mediating Env''s association with and targeting to Triton X-100-resistant lipid rafts.     

Cloning,high level expression and immunogenicity of 1163-1256 residues of C-terminal heavy chain of C. botulinum neurotoxin type E

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release from peripheral cholinergic synapses. BoNTs consist of a toxifying light chain and a heavy chain (HC) linked through a disulfide bond. In the present study we explored the immunogenicity and protective capability of the most effective part corresponding to 1163-1256 residues of botulinum type E neurotoxin HC gene. DNA encoding the 93 C-terminal amino acid of HC residues was synthesized with optimal codon usage for expression. These DNA fragments were ligated into a pLivSelect vector and subcloned into expression vector pET32a. Recombinant plasmids were then transformed into Escherichia coli strain BL21 DE3. The recombinant protein was purified by nickel affinity gel column chromatography. The HC1163-1256 was identified by antibodies raised against BoNT/E. HC1163-1256 was shown to bind with synaptotagmin and gangliosides, indicating that the expressed and purified HC1163-1256 protein retains a functionally active conformation. The immunization with recombinant protein induced a protection level in mice. The immunization with 2 μg of the recombinant protein induced a significant protection level in mice. In conclusion, availability of the recombinant protein provides an effective system to study the biochemical and physical interactions involved during BoNT binding to nerve cells and protection against its toxicity.     

Collagen synthesis is required for ascorbic acid-enhanced differentiation of mouse embryonic stem cells into cardiomyocytes

Ascorbic acid has been reported to promote the differentiation of embryonic stem (ES) cells into cardiomyocytes; however, the specific functions of ascorbic acid have not been defined. A stable form of ascorbic acid, namely, l-ascorbic acid 2-phosphate (A2-P), significantly enhanced cardiac differentiation; this was assessed by spontaneous beating of cardiomyocytes and expression of cardiac-specific markers obtained from mouse ES cells. This effect of ascorbic acid was observed only when A2-P was present during the early phase of differentiation. Treatment with two types of collagen synthesis inhibitors, l-2-azetidine carboxylic acid and cis-4-hydroxy-d-proline, significantly inhibited the A2-P-enhanced cardiac differentiation, whereas treatment with the antioxidant N-acetyl cysteine showed no effect. These findings demonstrated that ascorbic acid enhances differentiation of ES cells into cardiomyocytes through collagen synthesis and suggest its potential in the modification of cardiac differentiation of ES cells.     

Glial Activation and Segmental Upregulation of Interleukin-1β (IL-1β) in the Rat Spinal Cord after Surgical Incision

The present study investigated the expression patterns of glial cells and interleukin-1β (IL-1β) in the rat spinal cord after a surgical incision, which is closely related with clinical postoperative pain. Microglia and astrocytes became activated in the spinal cord following incision. Real-time polymerase chain reaction (PCR) and immunohistochemisty showed that IL-1β mRNA and protein level in the spinal cord was transiently upregulated after surgical incision. The increased IL-1β-immunoreactivity (IR) was mainly localized in neurons but not the activated microglia or astrocytes. Although obvious increase in IL-1β-IR could be observed in the lumbar segments of the spinal cord ipsilateral to a hind paw incision, significant upregulation of IL-1β was not detected in the lumbar segments following thoracic incision. The present study indicated that surgical incision could induce glial activation and segmental upregulation of IL-1β in the spinal cord. The activated glial cells and upregulated IL-1β, in turn, may be involved in the incision-induced pain hypersensitivity.     

First detection of Rickettsia in soft-bodied ticks associated with seabirds, Japan

Rickettsia was first detected in seabird soft-bodied ticks, Carios capensis and C. sawaii in Japan. According to sequence analysis, Rickettsia in Japan was identical to Rickettsia scc31 in C. capensis in the U.S.A. This suggested that an environmental circulation had consisted among microorganisms, ticks and long distance migratory seabirds around the Pacific Ocean.     

A computer-aided approach to compare the production economics of fed-batch and perfusion culture under uncertainty

Fed-batch and perfusion culture dominate mammalian cell culture production processes. In this paper, a decision-support tool was employed to evaluate the economic feasibility of both culture modes via a case study based upon the large-scale production of monoclonal antibodies. The trade-offs between the relative simplicity but higher start-up costs of fed-batch processes and the high productivity but higher chances of equipment failure of perfusion processes were analysed. Deterministic analysis showed that whilst there was an insignificant difference (3%) between the cost of goods per gram (COG/g) values, the perfusion option benefited from a 42% reduction in capital investment and a 12% higher projected net present value (NPV). When Monte Carlo simulations were used to account for uncertainties in titre and yield, as well as the risks of contamination and filter fouling, the frequency distributions for the output metrics revealed that neither process route offered the best of both NPV or product output. A product output criterion was formulated and the options that met the criterion were compared based on their reward/risk ratio. The perfusion option was no longer feasible as it failed to meet the product output criterion and the fed-batch option had a 100% higher reward/risk ratio. The tool indicated that in this particular case, the probabilities of contamination and fouling in the perfusion option need to be reduced from 10% to 3% for this option to have the higher reward/risk ratio. The case study highlighted the limitations of relying on deterministic analysis alone.     

The Wnt co-receptor LRP5 is essential for skeletal mechanotransduction but not for the anabolic bone response to parathyroid hormone treatment

The cell surface receptor, low-density lipoprotein receptor-related protein 5 (LRP5) is a key regulator of bone mass. Loss-of-function mutations in LRP5 cause the human skeletal disease osteoporosis-pseudoglioma syndrome, an autosomal recessive disorder characterized by severely reduced bone mass and strength. We investigated the role of LRP5 on bone strength using mice engineered with a loss-of-function mutation in the gene. We then tested whether the osteogenic response to mechanical loading was affected by the loss of Lrp5 signaling. Lrp5-null (Lrp5-/-) mice exhibited significantly lower bone mineral density and decreased strength. The osteogenic response to mechanical loading of the ulna was reduced by 88 to 99% in Lrp5-/- mice, yet osteoblast recruitment and/or activation at mechanically strained surfaces was normal. Subsequent experiments demonstrated an inability of Lrp5-/- osteoblasts to synthesize the bone matrix protein osteopontin after a mechanical stimulus. We then tested whether Lrp5-/- mice increased bone formation in response to intermittent parathyroid hormone (PTH), a known anabolic treatment. A 4-week course of intermittent PTH (40 microg/kg/day; 5 days/week) enhanced skeletal mass equally in Lrp5-/- and Lrp5+/+ mice, suggesting that the anabolic effects of PTH do not require Lrp5 signaling. We conclude that Lrp5 is critical for mechanotransduction in osteoblasts. Lrp5 is a mediator of mature osteoblast function following loading. Our data suggest an important component of the skeletal fragility phenotype in individuals affected with osteoporosis-pseudoglioma is inadequate processing of signals derived from mechanical stimulation and that PTH might be an effective treatment for improving bone mass in these patients.     

Negative regulation of condensin I by CK2-mediated phosphorylation

Condensin I, which plays an essential role in mitotic chromosome assembly and segregation in vivo, constrains positive supercoils into DNA in the presence of adenosine triphosphate in vitro. Condensin I is constitutively present in a phosphorylated form throughout the HeLa cell cycle, but the sites at which it is phosphorylated in interphase cells differ from those recognized by Cdc2 during mitosis. Immunodepletion, in vitro phosphorylation, and immunoblot analysis using a phospho-specific antibody suggested that the CK2 kinase is likely to be responsible for phosphorylation of condensin I during interphase. In contrast to the slight stimulatory effect of Cdc2-induced phosphorylation of condensin I on supercoiling, phosphorylation by CK2 reduced the supercoiling activity of condensin I. CK2-mediated phosphorylation of condensin I is spatially and temporally regulated in a manner different to that of Cdc2-mediated phosphorylation: CK2-dependent phosphorylation increases during interphase and decreases on chromosomes during mitosis. These findings are the first to demonstrate a negative regulatory mode for condensin I, a process that may influence chromatin structure during interphase and mitosis.