Generating In Vivo Cloning Vectors for Parallel Cloning of Large Gene Clusters by Homologous Recombination

A robust method for the in vivo cloning of large gene clusters was developed based on homologous recombination (HR), requiring only the transformation of PCR products into Escherichia coli cells harboring a receiver plasmid. Positive clones were selected by an acquired antibiotic resistance, which was activated by the recruitment of a short ribosome-binding site plus start codon sequence from the PCR products to the upstream position of a silent antibiotic resistance gene in receiver plasmids. This selection was highly stringent and thus the cloning efficiency of the GFPuv gene (size: 0.7 kb) was comparable to that of the conventional restriction-ligation method, reaching up to 4.3 × 10⁴ positive clones per μg of DNA. When we attempted parallel cloning of GFPuv fusion genes (size: 2.0 kb) and carotenoid biosynthesis pathway clusters (sizes: 4 kb, 6 kb, and 10 kb), the cloning efficiency was similarly high regardless of the DNA size, demonstrating that this would be useful for the cloning of large DNA sequences carrying multiple open reading frames. However, restriction analyses of the obtained plasmids showed that the selected cells may contain significant amounts of receiver plasmids without the inserts. To minimize the amount of empty plasmid in the positive selections, the sacB gene encoding a levansucrase was introduced as a counter selection marker in receiver plasmid as it converts sucrose to a toxic levan in the E. coli cells. Consequently, this method yielded completely homogeneous plasmids containing the inserts via the direct transformation of PCR products into E. coli cells.     

The Effect of Static Stretch on Elastin Degradation in Arteries

Previously we have shown that gradual changes in the structure of elastin during an elastase treatment can lead to important transition stages in the mechanical behavior of arteries [1]. However, in vivo arteries are constantly being loaded due to systolic and diastolic pressures and so understanding the effects of loading on the enzymatic degradation of elastin in arteries is important. With biaxial tensile testing, we measured the mechanical behavior of porcine thoracic aortas digested with a mild solution of purified elastase (5 U/mL) in the presence of a static stretch. Arterial mechanical properties and biochemical composition were analyzed to assess the effects of mechanical stretch on elastin degradation. As elastin is being removed, the dimensions of the artery increase by more than 20% in both the longitude and circumference directions. Elastin assays indicate a faster rate of degradation when stretch was present during the digestion. A simple exponential decay fitting confirms the time constant for digestion with stretch (0.11±0.04 h⁻¹) is almost twice that of digestion without stretch (0.069±0.028 h⁻¹). The transition from J-shaped to S-shaped stress vs. strain behavior in the longitudinal direction generally occurs when elastin content is reduced by about 60%. Multiphoton image analysis confirms the removal/fragmentation of elastin and also shows that the collagen fibers are closely intertwined with the elastin lamellae in the medial layer. After removal of elastin, the collagen fibers are no longer constrained and become disordered. Release of amorphous elastin during the fragmentation of the lamellae layers is observed and provides insights into the process of elastin degradation. Overall this study reveals several interesting microstructural changes in the extracellular matrix that could explain the resulting mechanical behavior of arteries with elastin degradation.     

Comparative Characteristics of Porous Bioceramics for an Osteogenic Response In Vitro and In Vivo

Porous calcium phosphate ceramics are used in orthopedic and craniofacial applications to treat bone loss, or in dental applications to replace missing teeth. The implantation of these materials, however, does not induce stem cell differentiation, so suitable additional materials such as porous calcium phosphate discs are needed to influence physicochemical responses or structural changes. Rabbit adipose-derived stem cells (ADSC) and mouse osteoblastic cells (MC3T3-E1) were evaluated in vitro by the MTT assay, semi-quantitative RT-PCR, and immunoblotting using cells cultured in medium supplemented with extracts from bioceramics, including calcium metaphosphate (CMP), hydroxyapatite (HA) and collagen-grafted HA (HA-col). In vivo evaluation of the bone forming capacity of these bioceramics in rat models using femur defects and intramuscular implants for 12 weeks was performed. Histological analysis showed that newly formed stromal-rich tissues were observed in all the implanted regions and that the implants showed positive immunoreaction against type I collagen and alkaline phosphatase (ALP). The intramuscular implant region, in particular, showed strong positive immunoreactivity for both type I collagen and ALP, which was further confirmed by mRNA expression and immunoblotting results, indicating that each bioceramic material enhanced osteogenesis stimulation. These results support our hypothesis that smart bioceramics can induce osteoconduction and osteoinduction in vivo, although mature bone formation, including lacunae, osteocytes, and mineralization, was not prominent until 12 weeks after implantation.     

Mutation at Intronic Repeats of the Ataxia-Telangiectasia Mutated (ATM) Gene and ATM Protein Loss in Primary Gastric Cancer with Microsatellite Instability

Ataxia-telangiectasia mutated (ATM) is a Ser/Thr protein kinase that plays a critical role in DNA damage-induced signaling and initiation of cell cycle checkpoint signaling in response to DNA-damaging agents such as ionizing radiation. We have previously reported the ATM protein loss by immunohistochemistry (IHC) in 16% of human gastric cancer (GC) tissue. We hypothesized that ATM gene intron mutations targeted by microsatellite instability (MSI) cause ATM protein loss in a subset of GC. We studied mononucleotide mutations at the intron of ATM gene, ATM IHC and MSI in GC. Ten human gastric cancer cell lines were studied for the ATM gene mutation at introns, RT-PCR, direct sequencing, and immunohistochemistry. GC tissues of 839 patients were analyzed for MSI and ATM IHC. Among them, 604 cases were analyzed for the ATM mutations at introns preceding exon 6, exon 10 and exon 20. Two human GC cell lines (SNU-1 and -638) showed ATM intron mutations, deletion in RT-PCR and direct sequencing, and ATM protein loss by IHC. The frequencies of ATM mutation, MSI, and ATM protein loss were 12.9% (78/604), 9.2% (81/882) and 15.2% (134/839), respectively. Analysis of associations among MSI, ATM gene mutation, and ATM protein loss revealed highly co-existing ATM gene alterations and MSI. ATM intron mutation and ATM protein loss were detected in 69.3% (52/75) and 53.3% (40/75) of MSI positive GC. MSI positivity and ATM protein loss were present in 68.4% (52/76) and 48.7% (37/76) of GC with ATM intron mutation. ATM mutation and ATM protein loss had characteristics of old age, distal location of tumor, large tumor size, and histologic intestinal type. Our study might be interpreted as that ATM gene mutation at intron might be targeted by MSI and lead to ATM protein loss in a selected group of GC.     

Enlarged Cavum Septum Pellucidum as a Neurodevelopmental Marker in Adolescent-Onset Opiate Dependence

Objective

Adolescent-onset exposure to highly addictive substances such as opiates may induce far-reaching deleterious effects on later mental and physical health. However, little is known about the neurodevelopmental basis for adolescent-onset opiate dependence. Here we examined whether having an abnormally large cavum septum pellucidum (CSP), a putative marker of limbic structural maldevelopment, is associated with opiate dependence particularly beginning in adolescence.

Method

The overall length of the CSP and the prevalence of abnormal enlargement of the CSP were assessed and compared in 65 opiate-dependent subjects (41 adolescent-onset opiate users and 24 adult-onset opiate users) and 67 healthy subjects.

Results

Opiate-dependent subjects showed a greater prevalence of abnormal CSP enlargement relative to healthy subjects (odds ratio [OR]=3.64, p=0.034). The overall CSP length of adolescent-onset opiate-dependent subjects was greater, as compared not only with healthy subjects (F_1,104=11.03, p=0.001) but also with those who began opiate use during adulthood (F_1,61=4.43, p=0.039).

Conclusions

The current findings provide the first evidence that abnormal CSP enlargement, which reflects limbic system dysgenesis of neurodevelopmental origin, may be linked to later development of opiate dependence. In addition, a greater CSP length, which indicates more severe limbic abnormalities, appears to confer higher risk for earlier onset of opiate use.     

Yu Ping Feng San,an Ancient Chinese Herbal Decoction Containing Astragali Radix,Atractylodis Macrocephalae Rhizoma and Saposhnikoviae Radix,Regulates the Release of Cytokines in Murine Macrophages

Yu Ping Feng San (YPFS), a Chinese herbal decoction, is composed of Astragali Radix (AR; Huangqi), Atractylodis Macrocephalae Rhizoma (AMR; Baizhu) and Saposhnikoviae Radix (SR; Fangfeng) in a weight ratio of 1∶2∶1. Clinically, YPFS has been widely used to regulate immune functions; however, the action mechanism of it is not known. Here, we addressed this issue by providing detail analyses of chemical and biological properties of YPFS. By using rapid resolution liquid chromatography coupled with mass spectrometry, fifteen chemicals deriving from different herbs of YPFS were determined, and which served as a control for the standardization of the herbal extract of YPFS. In general, the amounts of chosen chemical markers were higher in a preparation of YPFS as compared to that of single herb or two-herb compositions. In order to reveal the immune functions of YPFS, the standardized extract was applied onto cultured murine macrophages. The treatment of YPFS stimulated the mRNA and protein expressions of pro-inflammatory cytokines via activation of NF-κB by enhancing IκBα degradation. In contrast, the application of YPFS suppressed the expressions of pro-inflammatory cytokines significantly in the lipopolysaccharide (LPS)-induced chronic inflammation model. In addition, YPFS could up regulate the phagocytic activity in cultured macrophages. These results therefore supported the bi-directional immune-modulatory roles of YPFS in regulating the releases of cytokines from macrophages.