Boronated thymidine analogues for boron neutron capture therapy

Concise synthetic methods for synthesizing 3-carboranyl thymidine analogues (3CTAs) modified with cyclic and acyclic alcohols have been developed. The synthesis of these potential boron neutron capture therapy (BNCT) agents and their preliminary biological evaluation is described.     

A simple and economical short-oligonucleotide-based approach to shRNA generation

RNAi (RNA interference) has become a popular means of knocking down a specific gene in vivo. The most common approach involves the use of chemically synthesized short interfering RNAs (siRNAs), which are relatively easy and fast to use, but which are costly and have only transient effects. These limitations can be overcome by using short hairpin RNA (shRNA) expression vectors. However, current methods of generating shRNA expression vectors require either the synthesis of long (50-70 nt) costly oligonucleotides or multi-step processes. To overcome this drawback, we have developed a one-step short-oligonucleotides- based method with preparation costs of only 15% of those of the conventional methods used to obtain essentially the same DNA fragment encoding shRNA. Sequences containing 19 bases homologous to target genes were synthesized as 17- and 31-nt DNA oligonucleotides and used to construct shRNA expression vectors. Using these plasmids, we were able to effectively silence target genes. Because our method relies on the one-step ligation of short oligonucleotides, it is simple, less error-prone, and economical.     

Rapid upregulation ofDehyrin3 andDehydrin4 in response to dehydration is a characteristic of drought-tolerant genotypes in barley

The identification of molecular markers and marker-aided selection are essential to the efficient breeding of drought-tolerant plants. However, because that characteristic is controlled by many quantitative trait loci, such markers that can screen and trace desirable barley genotypes in a segregating population or germplasm have not yet been determined. Relative water content has been used to estimate drought tolerance in plants because it is highly correlated with the drought index of yield. To develop reliable gene-specific markers for identifying tolerant versus susceptible genotypes, we performed suppression subtractive hybridization to identify candidate genes. We used two domestic barley cultivars, one having the highest RWC (drought-tolerant ‘Chalbori’) and the other having the lowest (drought-susceptible ‘Daebaekbori’). In response to dehydration at the early seedling stage, rapid upregulation ofDehydrin3 (Dhn3) andDhn4 occurred in the drought-tolerant genotypes, but not in the susceptible ones. Similar results were obtained with mature plants growing under frequent drought stress in the greenhouse. In addition,Dhn3 andDhn4 conferred higher drought tolerance when they were over-expressed in transgenicArabidopsis. Thus, in addition to using assessments of RWC, we propose thatDhn3 andDhn4 expressions can serve as drought-induced gene-specific markers to determine drought-tolerant barley genotypes at the seedling stage.     

Clinical Value of Core Length in Contemporary Multicore Prostate Biopsy

Objectives

There is little data about the clinical value of core length for prostate biopsy (PBx). We investigated the clinical values of various clinicopathological biopsy-related parameters, including core length, in the contemporary multi-core PBx.

Patients and Methods

Medical records of 5,243 consecutive patients who received PBx at our institution were reviewed. Among them, 3,479 patients with prostate-specific antigen (PSA) ≤10ng/ml level who received transrectal ultrasound (TRUS)-guided multi (≥12)-core PBx at our institution were analyzed for prostate cancer (PCa). Gleason score upgrading (GSU) was analyzed in 339 patients who were diagnosed with low-risk PCa and received radical prostatectomy. Multivariate logistic regression analyses for PCa detection and prediction of GSU were performed.

Results

The mean age and PSA of the entire cohort were 63.5 years and 5.4ng/ml, respectively. The overall cancer detection rate was 28.5%. There was no statistical difference in core length between patients diagnosed with PCa and those without PCa (16.1 ± 1.8 vs 16.1 ± 1.9mm, P = 0.945). The core length was also not significantly different (16.4 ± 1.7 vs 16.4 ± 1.6mm, P = 0.889) between the GSU group and non-GSU group. Multivariate logistic regression analyses demonstrated that the core length of PBx did not affect PCa detection in TRUS-guided multi-core PBx (P = 0.923) and was not prognostic for GSU in patients with low-risk PCa (P = 0.356).

Conclusions

In patients undergoing contemporary multi-core PBx, core length may not have significant impact on PCa detection and also GSU following radical prostatectomy among low-risk PCa group.     

Thioredoxin overexpression in HT-1080 cells induced cellular senescence and sensitization to gamma radiation

An increment of thioredoxin-1 (TRX) is observed in many human primary cancers and appears to contribute to an increase of cell growth and a resistance to chemotherapy. On the contrary, when TRX was overexpressed in the HT-1080 fibrosarcoma cells, the cell growth was retarded and chromosomal polyploidy and cellular senescence were induced. TRX-overexpression made HT-1080 cells resistant to an oxidative stress caused by H2O2 or paraquat. But these cells were significantly sensitive to ionizing radiation, showing an abrogation of the G2 checkpoint. Their DNA contents were twice of the controls and they expressed typical senescence markers. Their expression levels of p53 and cyclin-dependent kinase inhibitors (CDKI) were about 2-3-fold higher than the control. Nevertheless, cyclin D1 and D3, which are negatively regulated by CDKIs, were also increased. Overall, in HT-1080 cells the TRX-overexpression created a state of cellular senescence caused by a simultaneous stimulation of the mitogen-activated pathways and an inhibition of the cyclin-dependent kinases, which is known as a hypermitogenic arrest.     

Type I interferon signaling regulates Ly6C(hi) monocytes and neutrophils during acute viral pneumonia in mice

Type I interferon (IFN-I) plays a critical role in the homeostasis of hematopoietic stem cells and influences neutrophil influx to the site of inflammation. IFN-I receptor knockout (Ifnar1 ^−/−) mice develop significant defects in the infiltration of Ly6C^hi monocytes in the lung after influenza infection (A/PR/8/34, H1N1). Ly6C^hi monocytes of wild-type (WT) mice are the main producers of MCP-1 while the alternatively generated Ly6C^int monocytes of Ifnar1 ^−/− mice mainly produce KC for neutrophil influx. As a consequence, Ifnar1 ^−/− mice recruit more neutrophils after influenza infection than do WT mice. Treatment of IFNAR1 blocking antibody on the WT bone marrow (BM) cells in vitro failed to differentiate into Ly6C^hi monocytes. By using BM chimeric mice (WT BM into Ifnar1 ^−/− and vice versa), we confirmed that IFN-I signaling in hematopoietic cells is required for the generation of Ly6C^hi monocytes. Of note, WT BM reconstituted Ifnar1 ^−/− chimeric mice with increased numbers of Ly6C^hi monocytes survived longer than influenza-infected Ifnar1 ^−/− mice. In contrast, WT mice that received Ifnar1 ^−/− BM cells with alternative Ly6C^int monocytes and increased numbers of neutrophils exhibited higher mortality rates than WT mice given WT BM cells. Collectively, these data suggest that IFN-I contributes to resistance of influenza infection by control of monocytes and neutrophils in the lung.     

6′-Mercaptoguanosine-resistance is related with purF gene encoding 5′-phosphoribosyl-1′-pyrophosphate amidotransferase in inosine-5′-monophosphate overproducing Brevibacterium ammoniagenes

From the gene library constructed with the chromosomal DNA of 6-mercaptoguanosine (MGS)-resistant strain Brevibacterium ammoniagenes IPR-1, a DNA fragment which conferred MGS-resistance to the wild-type strain B. ammoniagenes ATCC6872 was cloned. The purF gene encoding 5-phosphoribosyl-1-pyrophosphate amidotransferase was identified from this fragment and its nucleotide sequence was determined. Wild type purF gene was also cloned by polymerase chain reaction using chromosomal DNA of ATCC6872 as the template and its sequence was determined. Two nucleotides, 583 A and 1065 A, of MGS-resistant purF gene had been changed from 583 G and 1065 G by mutagenesis, respectively. Both changes at position 583 and 1065 were proved to be responsible for MGS-resistance by site-directed mutagenesis.     

A rice (Oryza sativa L.) MAP kinase gene, OsMAPK44, is involved in response to abiotic stresses

We have isolated and characterized a putative rice MAPK gene (designated OsMAPK44) encoding for a protein of 593 amino acids that has the MAPK family signature and phosphorylation activation motif, TDY. Alignment of the predicted amino acid sequences of OsMAPK44 showed high homology with other rice MAPKs. Under normal conditions, the OsMAPK44 gene is highly expressed in root tissues, but relatively less in leaf and stem tissues of the japonica type rice plant (O. sativa L. Donggin). mRNA expression of the gene is highly inducible by salt and drought treatment, but not by cold treatment. Moreover, the mRNA level of the OsMAPK44 is up-regulated by exogenously applied Abscisic acid (ABA) and H₂O₂. When we compared the OsMAPK44 gene expression level between a salt sensitive indica cultivar (IR64) and a salt resistant indica cultivar (Pokkali), they showed some difference in expression kinetics with the salt treatment. OsMAPK44 gene expression in Pokkali was slightly up-regulated within 30 min and then disappeared rapidly, while IR64 maintained its expression for 1 h following down-regulation. Under the salinity stress, OsMAPK44 overexpression transgenic rice plants showed less damage and greater ratio of potassium and sodium than OsMAPK44 suppressed transgenic lines did, suggesting that OsMAPK44 may have a role to prevent damages due to working for favorable ion balance in the presence of salinity.     

Osteogenic differentiation of human periosteal-derived cells in a three-dimensional collagen scaffold

This study examined the osteogenic differentiation of cultured human periosteal-derived cells grown in a three dimensional collagen-based scaffold. Periosteal explants with the appropriate dimensions were harvested from the mandible during surgical extraction of lower impacted third molar. Periosteal-derived cells were introduced into cell culture. After passage 3, the cells were divided into two groups and cultured for 28 days. In one group, the cells were cultured in two-dimensional culture dishes with osteogenic inductive medium containing dexamethasone, ascorbic acid, and β-glycerophosphate. In the other group, the cells were seeded onto a three-dimensional collagen scaffold and cultured under the same conditions. We examined the bioactivity of alkaline phosphatase (ALP), the RT-PCR analysis for ALP and osteocalcin, and measurements of the calcium content in the periosteal-derived cells of two groups. Periosteal-derived cells were successfully differentiated into osteoblasts in the collagen-based scaffold. The ALP activity in the periosteal-derived cells was appreciably higher in the three-dimensional collagen scaffolds than in the two-dimensional culture dishes. The levels of ALP and osteocalcin mRNA in the periosteal-derived cells was also higher in the three-dimensional collagen scaffolds than in the two-dimensional culture dishes. The calcium level in the periosteal-derived cells seeded onto three-dimensional collagen scaffolds showed a 5.92-fold increase on day 7, 3.28-fold increase on day 14, 4.15-fold increase on day 21, and 2.91-fold increase on day 28, respectively, compared with that observed in two-dimensional culture dishes. These results suggest that periosteal-derived cells have good osteogenic capacity in a three-dimensional collagen scaffold, which provides a suitable environment for the osteoblastic differentiation of these cells.