The precore sequence of hepatitis B virus is required for nuclear localization of the core protein

The cellular localization of the precore/core and core proteins was studied by immunofluorescence following transfection of 143 thymidine kinase-negative (TK ) and Hep-G2 cells with expression constructs containing wild-type (hepatitis B e antigen [HBeAg]-positive) and precore mutant (HBeAg-negative) sequences. Precore/core constructs with the wild-type phenotype result in strong nuclear staining, while, in contrast, constructs expressing core antigen alone have strong cytoplasmic staining. These differences in the pattern of immunofluorescence staining may be caused by expression of the precore/core protein, some of which may be translocated into the nucleus, following removal of the signal peptide. In vitro translation experiments showed that the main protein products obtained in the presence of microsomal membranes were the precore/core protein and a truncated product representing the same protein without its signal peptide. Core protein expression from the precore mutant constructs was very much reduced, indicating that translational re-initiation was not very efficient. The significance of the precore/core protein being present in the nucleus is not clear, but suggests that it may be important in the replicative cycle of the virus. Finally, HBeAg produced by some of the constructs could not be detected because amino acid substitutions affected antibody-binding epitopes.     

Lactic acid-mediated suppression of Helicobacter pylori by the oral administration of Lactobacillus salivarius as a probiotic in a gnotobiotic murine model

OBJECTIVES: We examined whether or not the lactobacilli administered to treat Helicobacter pylori (H. pylori) infection can suppress the colonization of H. pylori, and we also sought to elucidate the mechanism of such suppression. METHODS: We used an in vitro culture system and an H. pylori-infected gnotobiotic murine model. RESULTS: Among the lactobacillus species examined in vitro, Lactobacillus salivarius (L. salivarius) but not L. casei or L. acidophilus proved to be capable of producing a high amount of lactic acid and thus completely inhibiting the growth of H. pylori in a mixed culture. The validity of L. salivarius as a probiotic to suppress H. pylori and thus reduce the inflammatory response was again confirmed in vivo by using an H. pylori-infected gnotobiotic murine model. CONCLUSION: Based on our findings, L. salivarius was found to be a potentially effective probiotic against H. pylori.     

The importance of CD54 and CD86 costimulation in T cells stimulated with Candida albicans and Dermatophagoides farinae antigens in patients with atopic dermatitis

A number of studies have demonstrated an increased frequency of allergen-specific T cells producing increased amounts of interleukin-4 (IL-4) and IL-5, but little interferon-y in both the peripheral blood and skin lesions of patients with atopic dermatitis (AD). In this study, to further clarify the characteristics of T cells obtained from AD patients, we examined the dependency of the antigen-specific proliferation of peripheral blood mononuclear cells (PBMC) from AD patients on costimulatory molecules. The antigens used were Candida albicans and Dermatophagoides farinae, for which AD patients show increased levels of IgE antibodies. PBMC from control healthy donors stimulated with these antigens incorporated [3H]-thymidine much more than PBMC from AD patients. The addition of anti-CD54, -CD40, -CD80 and -CD86 monoclonal antibodies to the cultures showed that the PBMC required only CD54 and CD86 for stimulation with C. albicans, but required CD54, CD80 and CD86 for stimulation with D. farinae. Among these monoclonal antibodies, the anti-CD54 antibody suppressed the proliferative responses of most PBMC, most effectively followed by the anti-CD86 antibody. However, there were no significant differences in the requirement for costimulatory molecules of PBMC proliferation stimulated with C. albicans or D. farinae between AD patients and healthy donors. Since many studies have suggested that T-helper type 1 and T-helper type 2 immune responses are different in their dependency on CD80 or CD86 costimulation, our present results suggest that the allergen-specific T cells of AD patients are not completely shifted to a T-helper type 2 subset.     

Caffeine abolishes the ultraviolet-induced REV3 translesion replication pathway in mouse cells

When a replicative DNA polymerase stalls upon encountering a photoproduct on the template strand, it is relieved by other low-processivity polymerase(s), which insert nucleotide(s) opposite the lesion. Using an alkaline sucrose density gradient sedimentation technique, we previously classified this process termed UV-induced translesion replication (UV-TLS) into two types. In human cancer cells or xeroderma pigmentosum variant (XP-V) cells, UV-TLS was inhibited by caffeine or proteasome inhibitors. However, in normal human cells, the process was insensitive to these reagents. Reportedly, in yeast or mammalian cells, REV3 protein (a catalytic subunit of DNA polymerase ζ) is predominantly involved in the former type of TLS. Here, we studied UV-TLS in fibroblasts derived from the Rev3-knockout mouse embryo (Rev3KO-MEF). In the wild-type MEF, UV-TLS was slow (similar to that of human cancer cells or XP-V cells), and was abolished by caffeine or MG-262. In 2 cell lines of Rev3KO-MEF (Rev3(-/-)p53(-/-)), UV-TLS was not observed. In p53KO-MEF, which is a strict control for Rev3KO-MEF, the UV-TLS response was similar to that of the wild-type. Introduction of the Rev3 expression plasmid into Rev3KO-MEF restored the UV-TLS response in selected stable transformants. In some transformants, viability to UV was the same as that in the wild-type, and the death rate was increased by caffeine. Our findings indicate that REV3 is predominantly involved in UV-TLS in mouse cells, and that the REV3 translesion pathway is suppressed by caffeine or proteasome inhibitors.     

Growth yield of Rhodopseudomonas spheroides in light-anaerobic and dark-aerobic cultures

An experimental fact that the quantum yield, defined by moles of thiocyanate ion released per unit light energy absorbed in a photochemical reaction of Reinecke's salt solution, remains almost unchanged in a visible wavelength range from 400 to 700 nm was applied to the measurement of light energy absorbed by Rhodopseudomonas spheroides in a continuous culture vessel in situ. This technique of light energy assessment was deemed to be significant in the sense that not only the light energy that was transmitted but also that that was scattered in three dimensions could be assessed. Values of growth yield, Y_kj, for light-anaerobic and dark-aerobic cultivations of this microorganism were 0.012 g cell per kJ and 0.028 g cell per kJ, respectively. These values sound reasonable in the light of relevant data on Y_kj published by other workers on various chemo-organotrophic and/or photolithotrophic microbes.     

A convenient method to estimate the rate of heat evolution in fermentation

The estimation of heat of fermentation is discussed with reference to experimental data obtained in glucose-, ammonia- and oxygen-limited chemostat cultures of a specific bacterium and also to previous data published by other workers. Whatever the microbial or cultural conditions might be, the heat of fermentation can be easily estimated from TOD (total oxygen demand) measurement, accompanied by the concept of available electrons. If aerobic pathways are dominant in a particular fermentation, the specific rate of heat evolution, Q_H, can be calculated approximately from Q_H = Q_o2 (?δH_o^7ast;), where ? 7delta;H_o^* = 106 kcal/mol O₂, using only Q_o2 values without the need for either TOD or calorimetric determination.     

Simulation of water-bloom in a eutrophic lake—III. Modeling the vertical migration and growth of Microcystis aeruginosa

The rate of vertical migration (rising or sinking) of Microcystis aeruginosa colonies in water was represented by Stokes' law. The density, p_p of the colony, estimated conversely from observations on vertical migration rate by using Stokes' law, was shown as a function of gas vacuoles' fraction, V_f in algal cells.Referring to the experimental studies by previous workers on factors that the affect the value of V_f, gas vacuoles in the cells were assumed to collapse instantly, V_f decreasing to V_feq once V_f values exceed those of V_feq. The latter values of V_feq were defined from a cumulative and normal distribution of gas vacuoles that withstand the turgor pressure. P, Incidentally, the regeneration rate of gas vacuoles in the cells that were subjected to sonication, yielding V_f = 0 (non-vacuolate) was constant regardless of the post-sonication environment of light and/or dark.Taking for granted that there exist upper and lower limits of turgor pressures for a given algal cell, an equation on the rate of change in turgor presure of the cell was derived. Presentation of these rate equations is a prerequisite for modeling and simulating emergence and/or disappearance of the waterbloom in still waters of eutrophic lakes and/or ponds.     

Atrophy of White Adipose Tissue Accompanied with Decreased Insulin-Stimulated Glucose Uptake in Mice Lacking the Small GTPase Rac1 Specifically in Adipocytes

Insulin stimulates glucose uptake in adipose tissue and skeletal muscle by inducing plasma membrane translocation of the glucose transporter GLUT4. Although the small GTPase Rac1 is a key regulator downstream of phosphoinositide 3-kinase (PI3K) and the protein kinase Akt2 in skeletal muscle, it remains unclear whether Rac1 also regulates glucose uptake in white adipocytes. Herein, we investigated the physiological role of Rac1 in white adipocytes by employing adipocyte-specific rac1 knockout (adipo-rac1-KO) mice. Subcutaneous and epididymal white adipose tissues (WATs) in adipo-rac1-KO mice showed significant reductions in size and weight. Actually, white adipocytes lacking Rac1 were smaller than controls. Insulin-stimulated glucose uptake and GLUT4 translocation were abrogated in rac1-KO white adipocytes. On the other hand, GLUT4 translocation was augmented by constitutively activated PI3K or Akt2 in control, but not in rac1-KO, white adipocytes. Similarly, to skeletal muscle, the involvement of another small GTPase RalA downstream of Rac1 was demonstrated. In addition, mRNA levels of various lipogenic enzymes were down-regulated in rac1-KO white adipocytes. Collectively, these results suggest that Rac1 is implicated in insulin-dependent glucose uptake and lipogenesis in white adipocytes, and reduced insulin responsiveness due to the deficiency of Rac1 may be a likely explanation for atrophy of WATs.