Akane Ohta

Diverse regulation of sensory signaling by C. elegans nPKC-epsilon/eta TTX-4

Molecular and pharmacological studies in vitro suggest that protein kinase C (PKC) family members play important roles in intracellular signal transduction. Nevertheless, the in vivo roles of PKC are poorly understood. We show here that nPKC‐epsilon/eta TTX‐4 in the nematode Caenorhabditis elegans is required for the regulation of signal transduction in various sensory neurons for temperature, odor, taste, and high osmolality. Interestingly, the requirement for TTX‐4 differs in different sensory neurons. In AFD thermosensory neurons, gain or loss of TTX‐4 function inactivates or hyperactivates the neural activity, respectively, suggesting negative regulation of temperature sensation by TTX‐4. In contrast, TTX‐4 positively regulates the signal sensation of ASH nociceptive neurons. Moreover, in AWA and AWC olfactory neurons, TTX‐4 plays a partially redundant role with another nPKC, TPA‐1, to regulate olfactory signaling. These results suggest that C. elegans nPKCs regulate different sensory signaling in various sensory neurons. Thus, C. elegans provides an ideal model to reveal genetically novel components of nPKC‐mediated molecular pathways in sensory signaling.

Mitochondria and viruses.

Abstract Mitochondria are involved in a variety of cellular metabolic processes, and their functions are regulated by extrinsic and intrinsic stimuli including viruses. Recent studies have shown that mitochondria play a central role in the primary host defense mechanisms against viral infections, and a number of novel viral and mitochondrial proteins are involved in these processes. Some viral proteins localize in mitochondria and interact with mitochondrial proteins to regulate cellular responses. This review summarizes recent findings on the functions and roles of these molecules as well as mitochondrial responses to viral infections.

Discriminating between Varicella-Zoster Virus Vaccine and Wild-Type Strains by Loop-Mediated Isothermal Amplification

ABSTRACT The loop-mediated isothermal amplification (LAMP) method was developed to distinguish between the varicella-zoster virus (VZV) vaccine (vOka) strain and wild-type strains. Two single nucleotide polymorphisms (SNPs) (nucleotide [nt] 105705 for VR-1 VZV LAMP and nt 106262 for VR-2 VZV LAMP) located in the open reading frame 62 gene were selected as LAMP targets. Amplified vOka DNA demonstrated a typical ladder pattern; however, no LAMP product was detected in reactions performed with DNAs from other human herpesviruses by either VR-1 VZV LAMP or VR-2 VZV LAMP. This result was confirmed by a turbidity assay. The sensitivities of both VR-1 and VR-2 VZV LAMP determined by either the turbidity assay or agarose gel electrophoresis were 100 copies per reaction. To discriminate the vOka strain from wild-type strains, VR-1 and VR-2 VZV LAMP products were digested with the appropriate restriction enzymes (SacII for VR-1 LAMP and SmaΙ for VR-2 LAMP). The digested products were clearly different in the vOka strain and wild-type strains. To evaluate the utility of the LAMP methods for rapid differentiation, viral DNA (without DNA extraction) in swab samples was directly tested. Wild-type VZV DNA was detected in 20 swab samples by either VR-1 VZV LAMP or VR-2 VZV LAMP. Sequence analysis confirmed the expected SNPs in the LAMP products amplified from the vOka strain and the five wild-type strains.

Light and pheromone-sensing neurons regulates cold habituation through insulin signalling in Caenorhabditis elegans

Temperature is a critical environmental stimulus that has a strong impact on an organism’s biochemistry. Animals can respond to changes in ambient temperature through behaviour or altered physiology. However, how animals habituate to temperature is poorly understood. The nematode C. elegans stores temperature experiences and can induce temperature habituation-linked cold tolerance. Here we show that light and pheromone-sensing neurons (ASJ) regulate cold habituation through insulin signalling. Calcium imaging reveals that ASJ neurons respond to temperature. Cold habituation is abnormal in a mutant with impaired cGMP signalling in ASJ neurons. Insulin released from ASJ neurons is received by the intestine and neurons regulating gene expression for cold habituation. Thus, temperature sensation in a light and pheromone-sensing neuron produces a robust effect on insulin signalling that controls experience-dependent temperature habituation.

Development of a Human Herpesvirus 6 Species-Specific Immunoblotting Assay

ABSTRACT In order to assess the full spectrum of human herpesvirus 6A (HHV-6A)- and HHV-6B-associated diseases, we sought to develop an HHV-6 species-specific serological assay based on immunoblot analysis. The immunodominant proteins encoded by open reading frame U11, p100 for HHV-6A (strain U1102) and 101K for HHV-6B (strain Z29), were selected to generate virus species-specific antigens. Recombinant p100 and 101K were produced in a prokaryotic expression system. The expression of these proteins was confirmed by using anti-His tag and 101K-specific monoclonal antibodies. HHV-6 species-specific antibodies were detected by immunoblotting in patient sera. Eighty-seven serum samples obtained from various subjects were utilized to determine the reliability of the method for clinical use. Ten of twelve exanthem subitum convalescent-phase sera reacted exclusively with 101K, whereas none of twelve acute-phase sera reacted with either protein. Two of three sera collected from HHV-6A-infected patients reacted with p100 and 101K. Although all five acute and convalescent-phase sera obtained from transplant recipients reacted exclusively with 101K, two of six convalescent-phase sera obtained from patients with drug-induced hypersensitivity syndrome reacted with both p100 and 101K. Of 38 sera obtained from healthy adults, 31 were positive for 101K antibody, while 4 reacted with both proteins. However, PCR analysis of peripheral blood mononuclear cells and saliva from these subjects did not detect HHV-6A DNA. In conclusion, this novel serological assay based on immunoblot analysis using recombinant HHV-6A p100 and HHV-6B 101K allowed us to discriminate between HHV-6A- and HHV-6B-specific antibodies.

Loop-mediated isothermal amplification for discriminating between human herpesvirus 6 A and B

Genotyping of human herpesvirus 6 (HHV-6) is important clinically, particularly for the diagnosis of neurological diseases. The objective of this study was to establish a rapid HHV-6 genotyping method using the loop-mediated isothermal amplification (LAMP) method. An AccI site is located in the target sequence of HHV-6 B, but not in HHV-6 A. LAMP products were digested with the AccI enzyme and then separated by agarose gel electrophoresis to differentiate the digest pattern of the two variants. The fragment patterns were clearly different between HHV-6 A and B. In order to evaluate the reliability of this HHV-6 genotyping method for use in the clinical laboratory, serum samples from 20 patients with either primary HHV-6 infection or viral reactivation were collected and analyzed. HHV-6 DNA was amplified directly from the serum samples and all 20 LAMP products were positive for HHV-6 B.

Novel and Conserved Protein Macoilin Is Required for Diverse Neuronal Functions in Caenorhabditis elegans

Neural signals are processed in nervous systems of animals responding to variable environmental stimuli. This study shows that a novel and highly conserved protein, macoilin (MACO-1), plays an essential role in diverse neural functions in Caenorhabditis elegans. maco-1 mutants showed abnormal behaviors, including defective locomotion, thermotaxis, and chemotaxis. Expression of human macoilin in the C. elegans nervous system weakly rescued the abnormal thermotactic phenotype of the maco-1 mutants, suggesting that macoilin is functionally conserved across species. Abnormal thermotaxis may have been caused by impaired locomotion of maco-1 mutants. However, calcium imaging of AFD thermosensory neurons and AIY postsynaptic interneurons of maco-1 mutants suggest that macoilin is required for appropriate responses of AFD and AIY neurons to thermal stimuli. Studies on localization of MACO-1 showed that C. elegans and human macoilins are localized mainly to the rough endoplasmic reticulum. Our results suggest that macoilin is required for various neural events, such as the regulation of neuronal activity.

Recombinant human monoclonal antibodies to human cytomegalovirus glycoprotein B neutralize virus in a complement-dependent manner

Human antibodies specific for HCMV are currently considered as potential anti-HCMV therapeutic agents. In this study, we used a combinatorial human antibody library to isolate and characterize complete human monoclonal antibodies that effectively neutralize HCMV in a complement-dependent manner. One hundred and six clones were isolated in two independent screens using HCMV virions and recombinant glycoprotein B, gB654, as antigens. All of the clones recognized the same molecule gB and were classified into 14 groups based on the amino acid sequence of the V(H) region. Seven representative clones from these 14 groups had a strong gB654 binding affinity by surface plasmon resonance (SPR). A pairwise binding competition analysis suggested that there were three groups based on differences in the gB recognition sites. Although Fab fragments of the seven groups showed strong affinity for gB, none of the Fab fragments neutralized HCMV infectivity in vitro. In contrast, complete human IgG(1) antibodies of at least three groups neutralized HCMV in a complement-dependent manner. These data suggest that potent therapeutic antibodies can be obtained from a human antibody library, including most of the functional antibodies that mediate humoral immunity to the selected pathogen.

Herpes simplex virus type 1 UL14 tegument protein regulates intracellular compartmentalization of major tegument protein VP16

BackgroundHerpes simplex virus type 1 (HSV-1) has a complicated life-cycle, and its genome encodes many components that can modify the cellular environment to facilitate efficient viral replication. The protein UL14 is likely involved in viral maturation and egress (Cunningham C. et al), and it facilitates the nuclear translocation of viral capsids and the tegument protein VP16 during the immediate-early phase of infection (Yamauchi Y. et al, 2008). UL14 of herpes simplex virus type 2 exhibits multiple functions (Yamauchi Y. et al, 2001, 2002, 2003).MethodsTo better understand the function(s) of UL14, we generated VP16-GFP-incorporated UL14-mutant viruses with either single (K51M) or triple (R60A, R64A, E68D) amino acid substitutions in the heat shock protein (HSP)-like sequence of UL14. We observed the morphology of cells infected with UL14-null virus and amino acid-substituted UL14-mutant viruses at different time points after infection.ResultsUL14(3P)-VP16GFP and UL14D-VP16GFP (UL14-null) viruses caused similar defects with respect to growth kinetics, compartmentalization of tegument proteins, and cellular morphology in the late phase. Both the UL14D-VP16GFP and UL14(3P)-VP16GFP viruses led to the formation of an aggresome that incorporated some tegument proteins but did not include nuclear-egressed viral capsids.ConclusionsOur findings suggest that a cluster of charged residues within the HSP-like sequence of UL14 is important for the molecular chaperone-like functions of UL14, and this activity is required for the acquisition of functionality of VP16 and UL46. In addition, UL14 likely contributes to maintaining cellular homeostasis following infection, including cytoskeletal organization. However, direct interactions between UL14 and VP16, UL46, or other cellular or viral proteins remain unclear.

Studies on neuronal function of a novel and conserved protein TTX-8

We have previously reported impaired LTP of field excitatory postsynaptic potentials in the CA1 area of hippocampal slices obtained from neuropathic mice and its restoration after blockade of glycine uptake via glycine transporter 1 (GlyT1). In the present study, neuropathic mice prepared by partial sciatic nerve ligation exhibited congnitive dysfunction in the novel object recognition test, which was improved by systemic, but not intrathecal, administration of the GlyT1 inhibitor NFPS. In hippocampal slices obtained from neuropathic mice, NMDA receptor-mediated excitatory postsynaptic currents (NMDA-EPSCs) elicited in CA1 pyramidal cells were less potentiated by bath-applied glycine. After treating slices with either NFPS or astrocyte specific metabolic inhibitor fluoroacetate, glycine potentiated NMDAEPSCs equally in slices from neuropathic and sham-operated mice. Together, uptake of glycine via glial GlyT1 was increased in the hippocampus after peripheral nerve injury, which may cause LTP impairment and cognitive dysfunction.