Two Distinct Functional Patterns of Hepatitis C Virus (HCV)-Specific T Cell Responses in Seronegative,Aviremic Patients

In hepatitis C Virus (HCV) high-risk groups, HCV-specific T cell responses have been detected in seronegative, aviremic persons who have no evidence of HCV infection. Herein, we investigated functional profiles of HCV-specific T-cell responses in seronegative, aviremic patients of a HCV high-risk group. Seventy seven hemodialysis patients with chronic renal disease were analyzed by IFN-γ ELISpot assays, and eight of 71 (11.3%) seronegative, aviremic patients displayed HCV-specific T-cell responses. Their HCV-specific memory T cells were characterized by assessing cytokine polyfunctionality, known to provide antiviral protection. By intracellular staining of IFN-γ, TNF-α, IL-2 and MIP-1β, we identified two distinct populations in the seronegative, aviremic patients: polyfunctional responders and TNF-α-predominant responders. In further analysis, occult HCV infection was excluded as a cause of the HCV-specific T cell response via secondary nested RT-PCR of HCV RNA in peripheral blood mononuclear cell samples. HCV-specific T cells targeted multiple epitopes including non-structural proteins in a single patient, implying that their T cells might have been primed by HCV proteins synthesized within the host. We conclude that HCV-specific memory T cells of seronegative, aviremic patients arise from authentic HCV replication in the host, but not from current occult HCV infection. By functional pattern of HCV-specific T cells, there are two distinct populations in these patients: polyfunctional responders and TNF-α-predominant responders.     

TGF-beta2 stimulates cranial suture closure through activation of the Erk-MAPK pathway

Cranial sutures are important growth sites of the skull. During suture closure, the dura mater is one of the most important sources of various positive and negative regulatory signals. Previous results indicate that TGF-beta2 from dura mater strongly accelerates suture closure, however, its exact regulatory mechanism is still unclear. In this study, we confirmed that removal of dura mater in calvarial organ culture strongly accelerates sagittal suture closure and that this effect is further enhanced by TGF-beta2 treatment. TGF-beta2 stimulated cell proliferation in the MC3T3-E1 cell line. Similarly, it stimulated the proliferation of cells in the sutural space in calvarial organ culture. Furthermore, TGF-beta2-mediated enhanced cell proliferation and suture closure were almost completely inhibited by an Erk-MAPK blocker, PD98059. These results indicate that TGF-beta2-induced activation of Erk-MAPK is an important signaling component that stimulates cell proliferation to enrich osteoprogenitor cells, thereby promoting their differentiation into osteoblasts to achieve a rapid calvarial bone expansion.     

Adenoviral proteins mimic nutrient/growth signals to activate the mTOR pathway for viral replication

Like tumor cells, DNA viruses have had to evolve mechanisms that uncouple cellular replication from the many intra- and extracellular factors that normally control it. Here we show that adenovirus encodes two proteins that activate the mammalian target of rapamycin (mTOR) for viral replication, even under nutrient/growth factor-limiting conditions. E4-ORF1 mimics growth factor signaling by activating PI3-kinase, resulting in increased Rheb.GTP loading and mTOR activation. E4-ORF4 is redundant with glucose in stimulating mTOR, does not affect Rheb.GTP levels and is the major mechanism whereby adenovirus activates mTOR in quiescent primary cells. We demonstrate that mTOR is activated through a mechanism that is dependent on the E4-ORF4 protein phosphatase 2A-binding domain. We also show that mTOR activation is required for efficient S-phase entry, independently of E2F activation, in adenovirus-infected quiescent primary cells. These data reveal that adenovirus has evolved proteins that activate the mTOR pathway, irrespective of the cellular microenvironment, and which play a requisite role in viral replication.     

Spectral and thermodynamic properties of Ag(I), Au(III), Cd(II), Co(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(IV), and Zn(II) binding by methanobactin from Methylosinus trichosporium OB3b

Methanobactin (mb) is a novel chromopeptide that appears to function as the extracellular component of a copper acquisition system in methanotrophic bacteria. To examine this potential physiological role, and to distinguish it from iron binding siderophores, the spectral (UV–visible absorption, circular dichroism, fluorescence, and X-ray photoelectron) and thermodynamic properties of metal binding by mb were examined. In the absence of Cu(II) or Cu(I), mb will bind Ag(I), Au(III), Co(II), Cd(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(VI), or Zn(II), but not Ba(II), Ca(II), La(II), Mg(II), and Sr(II). The results suggest metals such as Ag(I), Au(III), Hg(II), Pb(II) and possibly U(VI) are bound by a mechanism similar to Cu, whereas the coordination of Co(II), Cd(II), Fe(III), Mn(II), Ni(II) and Zn(II) by mb differs from Cu(II). Consistent with its role as a copper-binding compound or chalkophore, the binding constants of all the metals examined were less than those observed with Cu(II) and copper displaced other metals except Ag(I) and Au(III) bound to mb. However, the binding of different metals by mb suggests that methanotrophic activity also may play a role in either the solubilization or immobilization of many metals in situ.     

Molecular and biochemical characterization of hemoglobinase, a cysteine proteinase, in Paragonimus westermani

The mammalian trematode Paragonimus westermani is a typical digenetic parasite, which can cause paragonimiasis in humans. Host tissues and blood cells are important sources of nutrients for development, growth and reproduction of P. westermani. In this study, a cDNA clone encoding a 47 kDa hemoglobinase of P. westermani was characterized by sequencing analysis, and its localization was investigated immunohistochemically. The phylogenetic tree prepared based on the hemoglobinase gene showed high homology with hemoglobinases of Fasciola hepatica and Schistosoma spp. Moreover, recombinant P. westermani hemoglobinase degradaded human hemoglobin at acidic pH (from 3.0 to 5.5) and its activity was almost completely inhibited by E-64, a cysteine proteinase inhibitor. Immunohistochemical studies showed that P. westermani hemoglobinase was localized in the epithelium of the adult worm intestine implying that the protein has a specific function. These observations suggest that hemoglobinase may act as a digestive enzyme for acquisition of nutrients from host hemoglobin. Further investigations may provide insights into hemoglobin catabolism in P. westermani.     

Cloning and localization of MCdef, a defensin from Manila clams (Ruditapes philippinarum)

A defensin-like peptide was previously detected in hemocytes of Manila clams (Ruditapes philippinarum). In the current study, we cloned and characterized this defensin, designated MCdef. Cloning produced a full-length gene sequence of 201 bp predicted to encode a 66-amino-acid precursor protein maturing to a 44-amino-acid residue. Amino acid sequence analysis showed that MCdef is similar to defensins from marine mollusks and ticks. Phylogenetic analysis suggested that MCdef is closely related to defensins from Mytilus galloprovincialis (Mediterranean mussel) and Crassostrea gigas (Pacific cupped oyster). The three-dimensional structure of MCdef was modeled using the solution structure of C. gigas defensin as a template. With the exception of three variable loop areas, the modeled structure of MCdef was identical to that of C. gigas defensin. MCdef antiserum was raised against a synthetic MCdef peptide and verified by Western blotting using recombinant MCdef. RT-PCR analysis demonstrated high levels of MCdef mRNA in hemocytes and adductor, foot, gill, mantle, palp, and siphon tissues of Vibrio tapetis-infected Manila clams, whereas in V. tapetis-uninfected Manila clams, the level of MCdef mRNA was low in adductor, palp, and siphon tissues and even lower in the other tested tissues. Immunohistochemical analysis revealed high MCdef expression was detected in the gill, the mantle, and the digestive tubules of the diverticulum of V. tapetis-infected Manila clams. Minimum inhibitory concentration (MIC) of the purified rMCdef was determined. MCdef showed highest activity against Streptococcus iniae and Staphylococcus aureus.     

Butyric acid production from red algae by a newly isolated <Emphasis Type="Italic">Clostridium</Emphasis> sp. S1

Objective

To produce butyric acid from red algae such as Gelidium amansii in which galactose is a main carbohydrate, microorganisms utilizing galactose and tolerating inhibitors in hydrolysis including levulinic acid and 5-hydroxymethylfurfural (HMF) are required.

Results

A newly isolated bacterium, Clostridium sp. S1 produced butyric acid not only from galactose as the sole carbon source but also from a mixture of galactose and glucose through simultaneous utilization. Notably, Clostridium sp. S1 produced butyric acid and a small amount of acetic acid with the butyrate:acetate ratio of 45.4:1 and it even converted acetate to butyric acid. Clostridium sp. S1 tolerated 0.5–2 g levulinic acid/l and recovered from HMF inhibition at 0.6–2.5 g/l, resulting in 85–92 % butyric acid concentration of the control culture. When acid-pretreated G. amansii hydrolysate was used, Clostridium sp. S1 produced 4.83 g butyric acid/l from 10 g galactose/l and 1 g glucose/l.

Conclusion

Clostridium sp. S1 produces butyric acid from red algae due to its characteristics in sugar utilization and tolerance to inhibitors, demonstrating its advantage as a red algae-utilizing microorganism.

     

NMR study on the interaction between RPA and DNA decamer containing cis-syn cyclobutane pyrimidine dimer in the presence of XPA: implication for damage verification and strand-specific dual incision in nucleotide excision repair

In mammalian cells, nucleotide excision repair (NER) is the major pathway for the removal of bulky DNA adducts. Many of the key NER proteins are members of the XP family (XPA, XPB, etc.), which was named on the basis of its association with the disorder xerodoma pigmentosum. Human replication protein A (RPA), the ubiquitous single-stranded DNA-binding protein, is another of the essential proteins for NER. RPA stimulates the interaction of XPA with damaged DNA by forming an RPA–XPA complex on damaged DNA sites. Binding of RPA to the undamaged DNA strand is most important during NER, because XPA, which directs the excision nucleases XPG and XPF, must bind to the damaged strand. In this study, nuclear magnetic resonance (NMR) spectroscopy was used to assess the binding of the tandem high affinity DNA-binding domains, RPA-AB, and of the isolated domain RPA-A, to normal DNA and damaged DNA containing the cyclobutane pyrimidine dimer (CPD) lesion. Both RPA-A and RPA-AB were found to bind non- specifically to both strands of normal and CPD- containing DNA duplexes. There were no differences observed when binding to normal DNA duplex was examined in the presence of the minimal DNA-binding domain of XPA (XPA-MBD). However, there is a drastic difference for CPD-damaged DNA duplex as both RPA-A and RPA-AB bind specifically to the undamaged strand. The strand-specific binding of RPA and XPA to the damaged duplex DNA shows that RPA and XPA play crucial roles in damage verification and guiding cleavage of damaged DNA during NER.     

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.