Comparison of real-time and nested PCR assays for detection of herpes simplex virus DNA

We performed a real-time PCR assay to detect herpes simplex virus (HSV) DNA, and compared it prospectively with a nested PCR assay in 164 clinical samples (109 cerebrospinal fluid and 55 sera) from patients suspected of having neonatal HSV infection or HSV encephalitis. In 25 of 164 samples, HSV DNA was detected by the nested PCR assay. All samples positive for HSV DNA in the nested PCR assay were also positive in the real-time PCR assay, and all but two samples negative for HSV DNA in the nested assay were negative in the real-time assay. The real-time PCR assay thus had a sensitivity of 100% and a specificity of 99%, when compared with the nested assay. Sequential assays in a case of disseminated HSV showed that a decrease in HSV DNA paralleled clinical improvement. Quantification of HSV DNA by real-time PCR was useful for diagnosing and monitoring patients with HSV encephalitis and neonatal HSV infection.     

Identification of promoters bound by c-Jun/ATF2 during rapid large-scale gene activation following genotoxic stress

The NH2-terminal Jun kinases (JNKs) function in diverse roles through phosphorylation and activation of AP-1 components including ATF2 and c-Jun. However, the genes that mediate these processes are poorly understood. A model phenotype characterized by rapid activation of Jun kinase and enhanced DNA repair following cisplatin treatment was examined using chromatin immunoprecipitation with antibodies against ATF2 and c-Jun or their phosphorylated forms and hybridization to promoter arrays. Following genotoxic stress, we identified 269 genes whose promoters are bound upon phosphorylation of ATF2 and c-Jun. Binding did not occur following treatment with transplatin or the JNK inhibitor SP600125 or JNK-specific siRNA. Of 89 known DNA repair genes represented on the array, 23 are specifically activated by cisplatin treatment within 3-6 hr. Thus, the genotoxic stress response occurs at least partly via activation of ATF2 and c-Jun, leading to large-scale coordinate gene expression dominated by genes of DNA repair.     

Insect cytokine growth-blocking peptide triggers a termination system of cellular immunity by inducing its binding protein

Growth-blocking peptide (GBP) is a 25-amino acid cytokine found in lepidopteran insects that possesses diverse biological activities such as stimulation of immune cells (plasmatocytes), cell proliferation, and larval growth regulation. We found another novel function of GBP that induces a hemolysis of another class of blood cells (oenocytoids). In the lysate of oenocytoids we identified a GBP-binding protein that shows a specific affinity for GBP. The characterization of purified GBP-binding protein and its cDNA demonstrated it as a 49.5-kDa novel protein with a C-terminal region displaying limited homology to several insect lipoproteins. Results of Northern and Western blotting indicated that the GBP-binding protein should be synthesized only in blood cells. Immunoelectron microscopic analyses confirmed that indirect immunoreactive signals were mostly localized in oenocytoids. Kinetic and biological analyses of interaction between GBP and the binding protein showed their strong binding was followed by clearance of GBP from hemolymph, thus indicating that this protein might function as an inhibitory factor against GBP. Based on these results, we propose that insect cytokine GBP shows multifunctions even in cellular immunity: it serves to stimulate immune cells and afterward silences its own action by inducing the binding protein through specific hemolysis.     

A novel method for study of protein folding kinetics by monitoring diffusion coefficient in time domain

Molecular diffusion process after the photo-induced electron injection to ferric cytochrome c (Fe(III) cyt c) in guanidine hydrochloride (GdnHCl) 3.5 M buffer solution is studied by the time-resolved transient grating technique. Circular dichroism studies have revealed that Fe(III) cyt c is unfolded under this condition but the reduced form, Fe(II) cyt c, is folded. Hence, this pulsed laser-induced reduction should initiate the folding process of cyt c. The observed transient grating signal shows prominent features, which have never been observed before. Based on several characteristic points, we concluded that the apparent diffusion coefficient (D) of Fe(II) cyt c after the reduction is time dependent, which must be associated with the protein folding dynamics. This time-dependent apparent D should reflect either the continuous time development of the hydrodynamic radius or population change of the unfolded and folded states during the folding dynamics. This is the first observation of the time-dependent apparent D during any chemical reaction, and this time-dependent measurement of D should be a unique and powerful way to study the protein folding kinetics from a viewpoint of the protein's shape or the protein-water intermolecular interaction.     

Developmentally regulated extrachromosomal circular DNA formation in the mesozoan <Emphasis Type="Italic">Dicyema japonicum</Emphasis>

The dicyemid mesozoans are simple multicellular parasites with a long cylindrical axial cell surrounded by a single outer layer of 20 to 30 ciliated peripheral somatic cells. Their larval development proceeds within the axial cell. Here we demonstrate the appearance of extrachromosomal circular DNAs and their fate during early embryogenesis in Dicyema japonicum. These DNAs are highly heterogeneous in sequence, suggesting that they consist of unique--not repetitive--elements. Potential open reading frames were not evident in the elements, so these DNAs are unlikely to have a protein-encoding function. In situ hybridization revealed that the circular DNA elements were restricted to the early embryonic larvae and gradually faded out as larvae approached maturity. Furthermore Southern blot analysis and polymerase chain reaction analysis using a high molecular weight DNA as a template provided evidence that the extrachromosomal DNA circles are originally present in chromosomes. These observations suggest DNA elimination--or selective replication--of the elements from chromosomes during early embryogenesis in dicyemid mesozoans.     

Cofilin phosphorylation and actin polymerization by NRK/NESK,a member of the germinal center kinase family

Nck-interacting kinase (NIK)-related kinase (NRK)/NIK-like embryo-specific kinase (NESK) is a protein kinase that belongs to the germinal center kinase family, and activates the c-Jun N-terminal kinase (JNK) signaling pathway. In this study, we examined the effect of NRK/NESK on actin cytoskeletal organization. Overexpression of NRK/NESK in COS7 cells induced accumulation of polymerized actin at the perinuclear. Phosphorylation of cofilin, an actin-depolymerizing factor, was increased in NRK/NESK-expressing HEK 293T cells. In addition, in vitro phosphorylation of cofilin was observed on NRK/NESK immunoprecipitates from HEK 293T cells expressing the kinase domain of NRK/NESK. The cofilin phosphorylation occurred at the serine residue of position 3 (Ser-3). Since the phosphorylation at Ser-3 inactivates the actin-depolymerizing activity of cofilin, these results suggest that NRK/NESK induces actin polymerization through cofilin phosphorylation. The cofilin phosphorylation did not appear to be mediated through activation of LIM-kinasel, a cofilin-phosphorylating kinase, or through the activation of JNK. Thus, cofilin is likely to be a direct substrate of NRK/NESK. NRK/NESK is predominantly expressed in skeletal muscle during the late stages of mouse embryogenesis. Thus, NRK/NESK may be involved in the regulation of actin cytoskeletal organization in skeletal muscle cells through cofilin phosphorylation.     

Tumor targeting of radiometal labeled anti-CEA recombinant T84.66 diabody and t84.66 minibody: comparison to radioiodinated fragments

Recombinant antibody fragments offer potential advantages over intact monoclonal antibodies in the radioimmunoscintigraphy (RIS) of solid tumors. Due to their smaller molecular size, antibody fragments have shown rapid tumor targeting and blood clearance, a more uniform tumor distribution and a lower potential to elicit a human immune response. Previously, we have expressed two genetically engineered antibody fragments, the T84.66 diabody (scFv dimer) and the T84.66 minibody (scFv-CH3 dimer), specific to carcinoembryonic antigen (CEA). When radioiodinated, both antibody fragments exhibited rapid tumor targeting and rapid blood clearance in xenografted mice. To extend and optimize their future clinical RIS utility with radiometals, these antibody fragments were conjugated with the macrocycle 1,4,7,10-tetraazacyclododecane N,N',N' ',N' "-tetraacetic acid (DOTA) and labeled with 111In. Tumor targeting and biodistribution studies were carried out in athymic mice xenografted with a human colorectal tumor cell line, LS174T. The [111In]T84.66 diabody (55 kDa) exhibited very rapid tumor targeting with 12.5 +/- 0.4% injected dose per gram (% ID g(-1) +/- standard error) at 2 h and reached a maximum of 13.3 +/- 0.9% ID g(-1) at 6 h. However, kidney uptake was observed to reached a peak of 183.5 +/- 21.0% ID g(-1) at 6 h, a result similar to that reported by others for other low molecular weight fragments labeled with radiometals. Preadministration of an oral dose of D-lysine resulted in a 59% lowering of the renal accumulation at 6 h, but was accompanied by a 31% reduction of tumor uptake to 9.2 +/- 1.2% ID g(-1). The second recombinant antibody fragment, the [111In]T84.66 minibody (80 kDa), displayed rapid tumor targeting of 14.2 +/- 6.1% ID g(-1) at 2 h, and reached a maximum activity of 24.5 +/- 6.1% ID g(-1) by 12 h. Renal uptake achieved a plateau of 12-13% ID g(-1) which cleared to 7.2% ID g(-1) at 72 h. However, hepatic uptake was elevated and reached a maximum of 26.0 +/- 1.0% ID g(-1) at 12 h in these xenograft-bearing mice. Experiments in nontumor bearing mice showed a reduction of hepatic activity at 12 h to 16.6 +/- 1.5% ID g(-1), indicative of an intrinsic hepatic accumulation of the [111In]DOTA-T84.66 minibody or metabolites. While the anti-CEA [111In]DOTA-T84.66 diabody and T84.66 minibody retain the rapid tumor targeting properties of the radioiodinated form, the normal organ accumulation (kidneys and liver, respectively) of the [111In]DOTA forms appeared problematic for RIS and RIT applications. Development of alternative blocking strategies or new metabolizable chelates are under investigation to enhance the utility of the radiometal form of these and other promising recombinant antibody fragments.     

A putative pheromone receptor gene is expressed in two distinct olfactory organs in goats

Mammals possess two anatomically and functionally distinct olfactory systems. The olfactory epithelium (OE) detects volatile odorants, while the vomeronasal organ (VNO) detects pheromones that elicit innate reproductive and social behavior within a species. In rodent VNO, three multigene families that encode the putative pheromone receptors, V1Rs, V2Rs and V3Rs, have been expressed. We have identified the V1R homologue genes from goat genomic DNA (gV1R genes). Deduced amino acid sequences of gV1R genes show 40-50% and 20-25% identity to those of rat and mouse V1R and V3R genes, respectively, suggesting that the newly isolated goat receptor genes are members of the V1R gene family. One gene (gV1R1 gene) has an open reading frame that encodes a polypeptide of 309 amino acids. It is expressed not only in VNO but also in OE. In situ hybridization analysis revealed that gV1R1 -expressing cells were localized in neuropithelial layers of VNO and OE. These results suggest that the goat may detect pheromone molecules through two distinct olfactory organs.