Mutational analysis of the length of the J3/4 domain of Escherichia coli ribonuclease P ribozyme

We prepared a series of length variants of the J3/4 domain of Escherichia coli ribonuclease P (RNase P) ribozyme: the four-base long J3/4 domain (A(62)G(63)G(64)A(65)) was replaced with GGA (denoted DeltaA), GA (DeltaAG), A (DeltaAGG), AAGGA (SigmaA), AAAGGA (SigmaAA), and AAAAGGA (SigmaAAA). The results indicated that truncating and inserting operations of the J3/4 domain drastically reduced ribozyme activity (WT>SigmaAA>SigmaA>SigmaAAA>DeltaAG>DeltaA, DeltaAGG), but did not affect the cleavage site selection of a substrate by the ribozyme. The reduced ribozyme activity of each mutant was rescued to some extent by the addition of a high concentration of magnesium ions. Our data indicate that the conserved AGGA sequence was important for efficient ribozyme reactions, and suggested that the length mutations affected ribozyme activity through metal ion binding steps.     

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.     

Targeted isolation of a designated region of the Bacillus subtilis genome by recombinational transfer

A method for positional cloning of the Bacillus subtilis genome was developed. The method requires a set of two small DNA fragments that flank the region to be copied. A 38-kb segment that carries genes ppsABCDE encoding five enzymes for antibiotic plipastatin synthesis and another genome locus as large as 100 kb including one essential gene were examined for positional cloning. The positional cloning vector for ppsABCDE was constructed using a B. subtilis low-copy-number plasmid that faithfully copied the precise length of the 38-kb DNA in vivo via the recombinational transfer system of this bacterium. Structure of the copied DNA was confirmed by restriction enzyme analyses. Furthermore, the unaltered structure of the 38-kb DNA was demonstrated by complementation of a ppsABCDE deletion mutant.     

Endothelial progenitor cells: past, state of the art, and future

Recent evidences suggest that endothelial progenitor cells (EPCs) derived from bone marrow (BM) contribute to de novo vessel formation in adults occurring as physiological and pathological responses. Emerging preclinical trials have shown that EPCs home to sites of neovascularization after ischemic events in limb and myocardium. On the basis of these aspects, EPCs are expected to develop as a key strategy of therapeutic applications for the ischemic organs. Such clinical requirements of EPCs will tentatively accelerate the translational research aiming at the devices to acquire the optimized quality and quantity of EPCs. In this review, we attempt to discuss about biological features of EPCs and speculate on the clinical potential of EPCs for therapeutic neovascularization.     

A Caenorhabditis elegans TGF-beta, DBL-1, controls the expression of LON-1, a PR-related protein, that regulates polyploidization and body length

Using cDNA-based array analysis combined with double-stranded RNA interference (dsRNAi), we have identified yk298h6 as a target gene of Caenorhabditis elegans TGF-beta signaling. Worms overexpressing dbl-1, a TGF-beta ligand, are 16% longer than wild type. Array analysis shows yk298h6 to be one of several genes suppressed in such worms. Disruption of yk298h6 function by dsRNAi also resulted in long worms, suggesting that it is a negative regulator of body length. yk298h6 was then mapped to, and shown to be identical to, lon-1, a known gene that affects body length. lon-1 encodes a 312 amino acid protein with a motif sequence that is conserved from plants to humans. Expression studies confirm that LON-1 is repressed by DBL-1, suggesting that LON-1 is a novel downstream component of the C.elegans TGF-beta growth regulation pathway. Consistent with this, LON-1 is expressed mainly in the larval and adult hypodermis and has dose-dependent effects on body length associated with changes in hypodermal ploidy, but not hypodermal cell proliferation.     

Comparative analyses of hairpin substrate recognition by Escherichia coli and Bacillus subtilis ribonuclease P ribozymes

Previously, we reported that the substrate shape recognition of the Escherichia coli ribonuclease (RNase) P ribozyme depends on the concentration of magnesium ion in vitro. We additionally examined the Bacillus subtilis RNase P ribozyme and found that the B. subtilis enzyme also required high magnesium ion, above 10 mM, for cleavage of a hairpin substrate. The results of kinetic studies showed that the metal ion concentration affected both the catalysis and the affinity of the ribozymes toward a hairpin RNA substrate.     

Functionality of insect‐cell‐derived colorectal cancer vaccine candidate protein EpCAM‐Fc in human dendritic cells

The baculovirus–insect cell expression system is widely used to produce recombinant proteins for various biomedical applications. Our previous study demonstrated that EpCAM, a colorectal cancer vaccine candidate protein, can be expressed in the baculovirus–insect cell expression system. However, its functionality (the ability to elicit an immune response), which is important for its possible use as a colorectal cancer vaccine for immunotherapy, still needed to be confirmed. In this study, we examined the ability of recombinant EpCAM to induce maturation of immature dendritic cells (DCs) derived from CD34⁺ cells isolated from human umbilical cord blood. We demonstrated that EpCAM induces the expression of four DC maturation markers: CD80, CD83, CD86 and MHC II. These results suggest that EpCAM produced in the baculovirus–insect cell expression system is functional in terms of its ability to trigger maturation of human DCs.