How environmental solution conditions determine the compaction velocity of single DNA molecules

Understanding the mechanisms of DNA compaction is becoming increasingly important for gene therapy and nanotechnology DNA applications. The kinetics of the compaction velocity of single DNA molecules was studied using two non-protein condensation systems, poly(ethylene glycol) (PEG) with Mg(2+) for the polymer-salt-induced condensation system and spermine for the polyamine condensation system. The compaction velocities of single tandem λ-DNA molecules were measured at various PEG and spermine concentrations by video fluorescent microscopy. Single DNA molecules were observed using a molecular stretching technique in the microfluidic flow. The results show that the compaction velocity of a single DNA molecule was proportional to the PEG or spermine concentration to the power of a half. Theoretical considerations indicate that the compaction velocity is related to differences in the free energy of a single DNA molecule between the random coil and compacted states. In the compaction kinetics with PEG, acceleration of the compaction velocity occurred above the overlap concentration while considerable deceleration occurred during the coexistence state of the random coil and the compacted conformation. This study demonstrates the control factors of DNA compaction kinetics and contributes toward the understanding of the compaction mechanisms of non-protein DNA interactions as well as DNA-protein interactions in vivo.     

Development of control serum for microbial antibody tests.

Control sera are an essential component of in-vitro clinical diagnostic reagents. We have developed a control serum for HBsAg, anti-HCV, anti-HIV and anti- Treponema pallidum testing. Since this control serum is prepared from human plasma, we paid special attention to viral safety. We incorporated three viral inactivation methods into the manufacturing process that maintain the necessary characteristics of a stable control serum.     

Sodium current function in adult and aged canine atrial cells

The incidence of atrial fibrillation increases with age, but it is unknown whether there are changes in the intrinsic function of Na+ currents in cells of the aged atria. Thus, we studied right (RA) and left (LA) atrial cells from two groups of dogs, adult and aged (>8 yr), to determine the change in Na+ currents with age. In this study all dogs were in normal sinus rhythm. Whole cell voltage clamp techniques were used to compare the Na+ currents in the two cell groups. Immunocytochemical studies were completed for the Na+ channel protein Na(v)1.5 to determine whether there was structural remodeling of this protein with age. In cells from aged animals, we found that Na+ currents are similar to those we measured in adult atria. However, Na+ current (I(Na)) density of the aged atria differed depending on the atrial chamber with LA cell currents being larger than RA cell currents. Thus with age, the difference in I(Na) density between atrial chambers remains. I(Na) kinetic differences between aged and adult cells included a significant acceleration into the inactivated state and an enhanced use-dependent decrease in peak current in aged RA cells. Finally, there is no structural remodeling of the cardiac Na+ channel protein Na(v)1.5 in the aged atrial cell. In conclusion, with age there is no change in I(Na) density, but there are subtle kinetic differences contributing to slight enhancement of use dependence. There is no structural remodeling of the fast Na+ current protein with age.     

Capillary affinity gel electrophoresis: new technique for specific recognition of DNA sequence and the mutation detection on DNA

The present state of studies on capillary affinity gel electrophoresis, which is a new technique for the specific recognition of a target DNA sequence, is reviewed. This article includes the principle, theory, methods, and applications of this technology. The great potential of capillary affinity gel electrophoresis for the sequence-specific recognition of DNA and the detection of mutations in specific genes is illustrated.     

Targeting of antigen to dendritic cells with poly(gamma-glutamic acid) nanoparticles induces antigen-specific humoral and cellular immunity

Nanoparticles are considered to be efficient tools for inducing potent immune responses by an Ag carrier. In this study, we examined the effect of Ag-carrying biodegradable poly(gamma-glutamic acid) (gamma-PGA) nanoparticles (NPs) on the induction of immune responses in mice. The NPs were efficiently taken up by dendritic cells (DCs) and subsequently localized in the lysosomal compartments. gamma-PGA NPs strongly induced cytokine production, up-regulation of costimulatory molecules, and the enhancement of T cell stimulatory capacity in DCs. These maturational changes of DCs involved the MyD88-mediated NF-kappaB signaling pathway. In vivo, gamma-PGA NPs were preferentially internalized by APCs (DCs and macrophages) and induced the production of IL-12p40 and IL-6. The immunization of mice with OVA-carrying NPs induced Ag-specific CTL activity and Ag-specific production of IFN-gamma in splenocytes as well as potent production of Ag-specific IgG1 and IgG2a Abs in serum. Furthermore, immunization with NPs carrying a CD8(+) T cell epitope peptide of Listeria monocytogenes significantly protected the infected mice from death. These results suggest that Ag-carrying gamma-PGA NPs are capable of inducing strong cellular and humoral immune responses and might be potentially useful as effective vaccine adjuvants for the therapy of infectious diseases.     

CD47(Low) Status on CD4 Effectors Is Necessary for the Contraction/Resolution of the Immune Response in Humans and Mice

How do effector CD4 T cells escape cell death during the contraction of the immune response (IR) remain largely unknown. CD47, through interactions with thrombospondin-1 (TSP-1) and SIRP-α, is implicated in cell death and phagocytosis of malignant cells. Here, we reported a reduction in SIRP-α-Fc binding to effector memory T cells (T(EM)) and in vitro TCR-activated human CD4 T cells that was linked to TSP-1/CD47-induced cell death. The reduced SIRP-α-Fc binding (CD47(low) status) was not detected when CD4 T cells were stained with two anti-CD47 mAbs, which recognize distinct epitopes. In contrast, increased SIRP-α-Fc binding (CD47(high) status) marked central memory T cells (T(CM)) as well as activated CD4 T cells exposed to IL-2, and correlated with resistance to TSP-1/CD47-mediated killing. Auto-aggressive CD4 effectors, which accumulated in lymph nodes and at mucosal sites of patients with Crohn's disease, displayed a CD47(high) status despite a high level of TSP-1 release in colonic tissues. In mice, CD47 (CD47(low) status) was required on antigen (Ag)-specific CD4 effectors for the contraction of the IR in vivo, as significantly lower numbers of Ag-specific CD47(+/+)CD4 T cells were recovered when compared to Ag-specific CD47(-/-) CD4 T cells. In conclusion, we demonstrate that a transient change in the status of CD47, i.e. from CD47(high) to CD47(low), on CD4 effectors regulates the decision-making process that leads to CD47-mediated cell death and contraction of the IR while maintenance of a CD47(high) status on tissue-destructive CD4 effectors prevents the resolution of the inflammatory response.     

The antennal system and cockroach evasive behavior. I. Roles for visual and mechanosensory cues in the response

Cockroaches escape from predators by turning and then running. This behavior can be elicited when stimuli deflect one of the rostrally located and highly mobile antennae. We analyzed the behavior of cockroaches, under free-ranging conditions with videography or tethered in a motion tracking system, to determine (1) how antennal positional dynamics influence escape turning, and (2) if visual cues have any influence on antennal mediated escape. The spatial orientation of the long antennal flagellum at the time of tactile stimulation affected the direction of resultant escape turns. However, the sign of flagellar displacement caused by touch stimuli, whether it was deflected medially or laterally for example, did not affect the directionality of turns. Responsiveness to touch stimuli, and escape turn performance, were not altered by blocking vision. However, because cockroaches first orient an antenna toward stimuli entering the peripheral visual field, turn direction can be indirectly influenced by visual input. Finally, when vision was blocked, the run phase of escape responses displayed reduced average velocities and distances traveled. Our results suggest that tactile and visual influences are integrated with previously known wind-sensory mechanisms to achieve multisensory control of the full escape response.     

Birth of mice produced by germ cell nuclear transfer

That mammals can be cloned by nuclear transfer indicates that it is possible to reprogram the somatic cell genome to support full development. However, the developmental plasticity of germ cells is difficult to assess because genomic imprinting, which is essential for normal fetal development, is being reset at this stage. The anomalous influence of imprinting is corroborated by the poor development of mouse clones produced from primordial germ cells (PGCs) during imprinting erasure at embryonic day 11.5 or later. However, this can also be interpreted to mean that, unlike somatic cells, the genome of differentiated germ cells cannot be fully reprogrammed. We used younger PGCs (day 10.5) and eventually obtained four full-term fetuses. DNA methylation analyses showed that only embryos exhibiting normal imprinting developed to term. Thus, germ cell differentiation is not an insurmountable barrier to cloning, and imprinting status is more important than the origin of the nucleus donor cell per se as a determinant of developmental plasticity following nuclear transfer.