Antigenic variation of the Epstein–Barr virus nuclear antigen EBNA1 as revealed by monoclonal antibodies

The Epstein–Barr virus (EBV) nuclear antigen EBNA1 plays an essential role in the replication of EBV episomes in latently infected cells and is the only viral protein that is consistently expressed in all programs of latent EBV gene expression. In this study, four monoclonal antibodies (MoAbs) directed to a region (amino acid residues 442–530) of EBNA1 were generated. Competitive enzyme-linked immunosorbent assay (ELISA) experiments using biotinylated MoAbs showed that they recognized distinct epitopes. Reactivity of these MoAbs with various laboratory EBV strains and field EBV isolates was shown to be heterogeneous in that EBNA1 from certain strains (isolates) was recognized and that from others was not. All four MoAbs showed such heterogeneous reactivity, and moreover, each MoAb showed a distinct spectrum of reactivity with these EBV strains (isolates). These results demonstrate an extensive structural variation in this region of EBNA1 as predicted by previous sequencing studies. These MoAbs will be useful as probes to dissect this structural heterogeneity of EBNA1.     

Detection and characterization of simian retroviruses homologous to human T-cell leukemia virus type I

Lymphoid cell lines were established from five different species of monkeys which were positive in antibodies cross-reactive with human T-cell leukemia virus type I (HTLV-I) and were shown to contain provirus sequences homologous to HTLV-I. Gene-specific probes of HTLV-I, gag, pol, env, pX, and LTR, hybridized efficiently with monkey DNAs from these cell lines under stringent conditions, indicating that the proviruses are very similar to HTLV-I along with whole viral genomes. However, the preliminary restriction mapping turned out the difference between simian retroviruses and HTLV-I and also among simian retroviruses. These findings suggest a common ancestor of simian and human retroviruses, but exclude the recent interspecies transmission between monkeys and humans.     

Computational studies on acquisition and adaptation of ocular following responses based on cerebellar synaptic plasticity

To investigate how cerebellar synaptic plasticity guides the acquisition and adaptation of ocular following response (OFR), a large-scale network model was developed. The model includes the cerebral medial superior temporal area (MST), Purkinje cells (P cells) of the ventral paraflocculus, the accessory optic and climbing fiber systems, the brain stem oculomotor network, and the oculomotor plant. The model reconstructed temporal profiles of both firing patterns of MST neurons and P cells and eye movements. Model MST neurons (n = 1,080) were set to be driven by retinal error and exhibited 12 preferred directions, 30 preferred velocities, and 3 firing waveforms. Correspondingly, each model P cell contained 1,080 excitatory synapses from granule cell axons (GCA) and 1,080 inhibitory synapses. P cells (n = 40) were classified into four groups by their laterality (hemisphere) and by preferred directions of their climbing fiber inputs (CF) (contralateral or upward). The brain stem neural circuit and the oculomotor plant were modeled on the work of Yamamoto et al. The initial synaptic weights on the P cells were set randomly. At the beginning, P cell simple spikes were not well modulated by visual motion, and the eye was moved only slightly by the accessory optic system. The synaptic weights were updated according to integral-differential equation models of physiologically demonstrated synaptic plasticity: long-term depression and long-term potentiation for GCA synapses and rebound potentiation for inhibitory synapses. We assumed that maximum plasticity was induced when GCA inputs preceded CF inputs by 200 ms. After more than 10,000 presentations of ramp-step visual motion, the strengths of both the excitatory and inhibitory synapses were modified. Subsequently, the simple spike responses became well developed, and ordinary OFRs were acquired. The preferred directions of simple spikes became the opposite of those of CFs. Although the model MST neurons were set to possess a wide variety of firing characteristics, the model P cells acquired only downward or ipsilateral preferred directions, high preferred velocities and stereotypical firing waveforms. Therefore the drastic transition of the neural representation from the population codes in the MST to the firing-rate codes of simple spikes were learned at the GCA-P cell synapses and inhibitory cells-P cell synapses. Furthermore, the model successfully reproduced the gain- and directional-adaptation of OFR, which was demonstrated by manipulating the velocity and direction of visual motion, respectively. When we assumed that synaptic plasticity could only occur if CF inputs preceded GCA inputs, the ordinary OFR were acquired but neither the gain-adaptation nor the directional adaptation could be reproduced.     

Immune responses against a single CD8+-T-cell epitope induced by virus vector vaccination can successfully control Trypanosoma cruzi infection

In order to develop CD8+-T-cell-mediated immunotherapy against intracellular infectious agents, vaccination using recombinant virus vectors has become a promising strategy. In this study, we generated recombinant adenoviral and vaccinia virus vectors expressing a single CD8+-T-cell epitope, ANYNFTLV, which is derived from a Trypanosoma cruzi antigen. Immunogenicity of these two recombinant virus vectors was confirmed by the detection of ANYNFTLV-specific CD8+ T cells in the spleens of immunized mice. Priming/boosting immunization using combinations of these two recombinant virus vectors revealed that the adenovirus vector was efficient for priming and the vaccinia virus vector was effective for boosting the CD8+-T-cell responses. Moreover, we also demonstrated that the ANYNFTLV-specific CD8+-T-cell responses were further augmented by coadministration of recombinant vaccinia virus vector expressing the receptor activator of NFkappaB (RANK) ligand as an adjuvant. By priming with the adenovirus vector expressing ANYNFTLV and boosting with the vaccinia virus vectors expressing ANYNFTLV and RANK ligand, the immunized mice were efficiently protected from subsequent challenge with lethal doses of T. cruzi. These results indicated, for the first time, that the induction of immune responses against a single CD8+-T-cell epitope derived from an intrinsic T. cruzi antigen was sufficient to control lethal T. cruzi infection.     

Total sleep deprivation elevates blood pressure through arterial baroreflex resetting: a study with microneurographic technique

STUDY OBJECTIVES: Sleep deprivation has a profound effect on cardiovascular regulation through the autonomic nervous system. This study examined the effect of 24-hour total sleep deprivation on muscle sympathetic nerve activity (MSNA), which is a direct measurement of the postganglionic sympathetic efferent innervating the vascular bed in the skeletal muscle and other circulatory structures. DESIGN: The study was performed on 6 young healthy men. The factors exerting influence on MSNA, such as aging, obesity, body posture, activity, intensity of illumination, and food and beverage consumption were strictly controlled. Burst rate and burst incidence were used as parameters of MSNA. The burst rate, burst incidence, heart rate, and systolic and diastolic blood pressure were measured after total sleep deprivation and control sleep. To perform a linear regression analysis of arterial baroreflex (ABR), the incidence of MSNA bursts corresponding to a given diastolic blood pressure (%MSNA) was examined. MEASUREMENT AND RESULTS: The diastolic blood pressure was significantly higher after total sleep deprivation than after control sleep (66.5 +/- 1.7 vs 57.4 +/- 3.3 mm Hg). The burst rate (9.6 +/- 1.8 vs 13.3 +/- 2.7 bursts/min) and burst incidence (21.6 +/- 4.5 vs 30.3 +/- 8.9 bursts/100 heart beats) of MSNA were significantly lower after total sleep deprivation than after control sleep (P < .05). Analysis of the ABR disclosed a significant linear regressive relation between %MSNA and diastolic blood pressure in every subject after both total sleep deprivation and control sleep. This result implies that the ABR regulates the occurrence of MSNA bursts under different diastolic blood pressure conditions. The threshold (X-axis intercept) of the blood pressure regression line (ie, an indicator of the ABR set point) shifted by 12 +/- 4.3 mm Hg toward a higher blood pressure level after total sleep deprivation (P < .05). The ABR sensitivity, or the slope of the regression line, tended to be less steep after total sleep deprivation than after control sleep, although it was not statistically significant (P = .09). CONCLUSIONS: The diastolic blood pressure increased and both burst rate and burst incidence of MSNA decreased after total sleep deprivation. The results show that resetting of the ABR toward a higher blood pressure level occurred after total sleep deprivation. This ABR resetting probably brings about an increase in arterial blood pressure after total sleep deprivation.     

Characterization of DNA sequences constituting the terminal heterochromatin of the chicken Z chromosome

Two clones, pCZTH5-8 and pCZTH12-8, were isolated from a female chicken genomic library by screening with sequences obtained from genomic libraries which had been constructed from a terminal region of a single Z chromosome of chicken utilizing laser microbeam irradiation and PCR amplification. Fluorescencein situ hybridization to the mitotic Z chromosome and the lampbrush ZW bivalent of chicken demonstrated that both the cloned sequences are located in the heterochromatic region of the Z chromosome at the end opposite to the pairing region with the W chromosome. The sequences pCZTH5-8 and pCZTH12-8 are distributed widely on both the telomeric bow-like loops (TBL) and the region I (short loops region) of the Z lampbrush chromosome. These clones, pCZTH12-8 particularly notably, hybridized also to the TBLs of lampbrush bivalents 1–4 of chicken. Both sequence are transcribed in the lampbrush stage oocytes on the Z chromosome and on other macrobivalents. The subfragment of pCZTH5-8 which hybridizes to the TBLs and the insert of pCZTH12-8 contain regions that are closely similar in sequence. The pCZTH5-8 sequence has no internal repeats and may be part of the 24-kb macrosatellite repeating unit that is evident afterNhel digestion of the genomic DNA. A cloned 24-kb unit, pFN-1, does not show significant DNA curvature, but cytosines of its CpG dinucleotides may be highly methylatedin vivo. This contrasts with the repeat sequences of the W heterochromatin which not only have highly methylated CpG but are also strongly curved. The 24-kb unit is repeated about 830 times in the diploid genome of a female chicken, suggesting that nearly the entire terminal heterochromatin on the Z chromosome consists of this macrosatellite family. Sequences of the greater part of the pCZTH5-8 are restricted to the genusGallus but the sequence of one subregion which hybridizes to TBLs is present in the genomes of the order Galliformes.accepted for publication by M. Schmid     

Ultrastructural observations on the vincristine-induced neuronal crystalloid inclusion in young rats

Summary Vincristine-induced crystalloid inclusions were examined in the neurons and neuronal processes of young rats by the electron microscope (EM) equipped with a tilting stage. Using a computer system that reproduces three-dimensional organization, an optical transformation method was applied to the microtubules and neurofilaments in an attempt to clarify the morphological appearance and internal pattern of crystalloid inclusions. The dimensional models obtained were compared with actual EM photographs, and the characteristic ultrastructural component and morphology were drawn out.Basically, a crystalloid inclusion is composed of four strands of intermediate 10 nm neurofilaments connected to one another by four side-arms producing a circular profile on a transverse section. These four side-arms seemed to arise from nodules within the filaments at regular intervals simulating a bead-like appearance. These data did not significantly differ from those obtained from EM images. Characteristically, these crystalloid inclusions began to appear 6 h after the administration of 10^–3 M vincristine sulfate and persisted up to a period of 6 days. Beyond that, however, these inclusions were no longer demonstrable suggesting a transient state. This was in contrast to neurofibrillary tangles which appeared to be permanent changes.     

Acute Low-Intensity Treadmill Running Upregulates the Expression of Intestinal Glucose Transporters via GLP-2 in Mice

The effects of exercise on nutrient digestion and absorption in the intestinal tract are not well understood. A few studies have reported that exercise training increases the expression of molecules involved in carbohydrate digestion and absorption. Exercise was also shown to increase the blood concentration of glucagon-like peptide-2 (GLP-2), which regulates carbohydrate digestion and absorption in the small intestine. Therefore, we investigated the effects of exercise on the expression of molecules involved in intestinal digestion and absorption, including GLP-2. Six-week-old male mice were divided into a sedentary (SED) and low-intensity exercise (LEx) group. LEx mice were required to run on a treadmill (12.5 m/min, 1 h), whereas SED mice rested. All mice were euthanized 1 h after exercise or rest, and plasma, jejunum, ileum, and colon samples were collected, followed by analysis via IHC, EIA, and immunoblotting. The levels of plasma GLP-2 and the jejunum expression of the GLP-2 receptor, sucrase-isomaltase (SI), and glucose transporter 2 (GLUT2) were higher in LEx mice. Thus, we showed that acute low-intensity exercise affects the expression of molecules involved in intestinal carbohydrate digestion and absorption via GLP-2. Our results suggest that exercise might be beneficial for small intestine function in individuals with intestinal frailty.     

Diagnostic impact of monitoring transcranial motor-evoked potentials to prevent ischemic complications during endovascular treatment for intracranial aneurysms

Neurosurgical Review - The present study aimed to determine the incidence of intraprocedural motor-evoked potential (MEP) changes and to correlate them with intraprocedural ischemic complications...     

All-in-One Silk Fibroin Sponge as the Vitrification Cryodevice of Rat Pancreatic Islets and the VEGF-Embedded Scaffold for Subrenal Transplantation

Islet transplantation is a promising option for the clinical treatment of insulin-dependent diabetes, but a reliable islet cryopreservation/transplantation protocol should be established to overcome the donor shortage. The current study reports that a silk fibroin (SF) sponge disk can be used as a cryodevice for vitrification of large quantity pancreatic islets and the scaffold for subsequent subrenal transplantation in a rat model. The marginal islet mass (550 islet equivalents [IEQs]) on an SF sponge disk was vitrified-warmed and transplanted beneath the kidney capsule of a streptozotocin-induced diabetic rat with or without vascular endothelial growth factor (VEGF). Subrenal transplantation (no scaffold) of 550 IEQ fresh islets and post-warm islets vitrified on a nylon mesh device resulted in achieving euglycemia of recipient rats at 60% and 0%, respectively. Transplantation of 550 IEQ islets vitrified-warmed on an SF sponge disk failed to achieve euglycemia of recipient rats (0%), but the VEGF inclusion in the SF sponge disk contributed to acquiring the euglycemic recipients (33%). All cured recipient rats regained hyperglycemia after nephrectomy, and the histopathologic analysis exhibited a well-developing blood vessel network into the islet engrafts. Thus, an SF sponge disc was successively available as the cryodevice for islet vitrification, the transporter of the angiogenic VEGF, and the scaffold for subrenal transplantation in the rat model.