Vasoactive-intestinal-polypeptide (VIP)-like immunoreactive cells in the skull base of rats

Summary We investigated the distribution of vasoactive intestinal polypeptide (VIP)-like immunoreactive cell bodies in relation to the major cerebral and internal carotid arteries at the skull base in rats. Acetylcholinesterase (AChE) histochemistry was also applied to investigate the localization of this enzyme. VIP staining revealed a few positive cell bodies in nerves close to the internal carotid artery at the base of the skull as well as in the cerebral arterial wall. Ganglion-like cell bodies were detectable within the greater superficial petrosal (GSP) nerve. AChE activity was observed in VIP-like immunoreactive cell bodies along the whole of the GSP nerve. These cell bodies in the GSP nerve may give rise to at least some of the perivascular VIP-and AChE-containing nerves of the internal carotid arteries at the base of the skull.     

Gene delivery to embryonic stem cells

Since the establishment of embryonic stem (ES) cells and the identification of tissue-specific stem cells, researchers have made great strides in the analysis of the natural biology of such stem cells for the development of therapeutic applications. Specifically, ES cells are capable of differentiating into all of the cell types that constitute the whole body. Thus, ES cell research promises new type of treatments and possible cures for a variety of debilitating diseases and injuries. The potential medical benefits obtained from stem cell technology are compelling and stem cell research sees a bright future. Control of the growth and differentiation of stem cells is a critical tool in the fields of regenerative medicine, tissue engineering, drug discovery, and toxicity testing. Toward such a goal, we present here an overview of gene delivery in ES cells, covering the following topics: significance of gene delivery in ES cells, stable versus transient gene delivery, cytotoxicity, suspension versus adherent cells, expertise, time, cost, viral vectors for gene transduction (lentiviruses, adenoviruses, and adeno-associated viruses, chemical methods for gene delivery, and mechanical or physical gene delivery methods (electroporation, nucleofection, microinjection, and nuclear transfer).     

Improved synthesis with high yield and increased molecular weight of poly(alpha,beta-malic acid) by direct polycondensation

The development of synthetic biodegradable polymers, such as poly(alpha-hydroxy acid), is particularly important for constructing medical devices, including scaffolds and sutures, and has attracted growing interest in the biomedical field. Here, we report a novel approach to preparing high molecular weight poly(malic acid) (HMW--PMA) as a biodegradable and bioabsorbable water-soluble polymer. We investigated in detail the reaction conditions for the simple direct polycondensation of l-malic acid, including the reaction times, temperatures, and catalysts. The molecular weight of synthesized alpha,beta-PMA is dependent on both the reaction temperature and time. The optimum reaction condition to obtain alpha,beta-PMA by direct polycondensation using tin(II) chloride as a catalyst was thus determined to be 110 degrees C for 45 h with a molecular weight of 5300. The method for alpha,beta-PMA synthesis established here will facilitate production of alpha,beta-PMA of various molecular weights, which may have a potential utility as biomaterials.     

Cancer Cell Analyses at the Single Cell-Level Using Electroactive Microwell Array Device

Circulating tumor cells (CTCs), shed from primary tumors and disseminated into peripheral blood, are playing a major role in metastasis. Even after isolation of CTCs from blood, the target cells are mixed with a population of other cell types. Here, we propose a new method for analyses of cell mixture at the single-cell level using a microfluidic device that contains arrayed electroactive microwells. Dielectrophoretic (DEP) force, induced by the electrodes patterned on the bottom surface of the microwells, allows efficient trapping and stable positioning of single cells for high-throughput biochemical analyses. We demonstrated that various on-chip analyses including immunostaining, viability/apoptosis assay and fluorescent in situ hybridization (FISH) at the single-cell level could be conducted just by applying specific reagents for each assay. Our simple method should greatly help discrimination and analysis of rare cancer cells among a population of blood cells.     

Zinc and copper accumulation and isometallothionein induction in mouse ascites sarcoma S180A cells.

To investigate Zn and Cu accumulation and isometallothionein (iso-MT) induction in ascites-sarcoma S180A cells, 5 micrograms of Zn2+ or Cu2+/g body weight was administered to tumour-bearing mice intraperitoneally. In the tumour cells the Zn or Cu concentration increased more than in the host liver, which is the target organ for those metals; the maximum Zn or Cu level was about 2-3 times that in the host liver. The amounts of Zn-MT or Cu-MT accumulated in the tumour cells and host liver were proportional to such dose accumulation levels in the each cytosol; the maximum level of Zn-MT or Cu-MT was 4 or 2 times higher than in the host liver. MT accumulated in the tumour cells showed two subfractions (MT-1 and MT-2); the ratio of Zn (or Cu) bound to MT-1 to that bound to MT-2 in the host liver and tumour cells was 1.0 (or 1.0) and 0.7 (or 0.25) respectively, suggesting that the induction level of MT-2 in the tumour cells is more than that of MT-1. The h.p.l.c. profiles (using an anion-exchange column) of the isolated MT-1 and MT-2 subfractions from Zn-treated normal-mouse liver showed a single peak (MT-1-1) and two peaks (MT-2-1 and MT-2-2) respectively; mouse MTs were separated into three isoforms. In the ascites cells, the MT fraction obtained by a gel filtration was also separated into three isoforms; however, the amount of MT-2-1 isoform was 3 times that in the Zn-treated normal-mouse liver.     

Polymorphisms in the promoter region of the porcine antiviral MX1 and MX2 genes

The porcine MX1 and MX2 promoters were characterized in this study. Sequencing of the 332-bp MX1 promoter region identified 15 substitutions and insertions at three positions in 21 pigs from 15 breeds, in which nine genotypes were classified. Among the nine genotypes, no statistically significant differences in the promoter activities were observed after interferon (IFN- α 2b) treatment of transiently transfected cells containing constructs with luciferase reporter plasmids. The 341-bp MX2 promoter region contained regulatory sequences for ISRE, GC box, Sp1 and AP-1, as well as a TATA box. Nucleotide sequences of the MX2 promoter region revealed four substitutions and one deletion, in which six genotypes were classified. Among the six genotypes, a statistically significant difference ( P  < 0.05) in MX2 promoter activities after IFN- α 2b treatment was detected in transiently transfected cells.     

Effect of indomethacin, tiaprofenic acid and dicrofenac on rat gastric mucosal damage and content of prostacyclin and prostaglandin E2

Gastric ulcerogenicity and depletion of endogenous prostaglandins (PGs) content induced by tiaprofenic acid, dicrofenac and indomethacin were examined using the same antiinflammatory effective doses. Male Wistar rats were given each of these drugs intragastrically 24, 18, and 3 hrs before sacrifice in the following doses (mg/kg): indomethacin (0.8, 4 and 20); tiaprofenic acid (1.2, 6 and 30); dicrofenac (0.8, 4 and 20). Endogenous prostacyclin (PGI2) and PGE2 in fundic mucosa were determined by radioimmunoassay. The three compounds produced fundic mucosal lesions in a dose-dependent manner. However, tiaprofenic acid and dicrofenac were both less potent than indomethacin in producing gastric mucosal lesions at similar antiinflammatory doses. Mucosal PGE2 content was abolished by the three compounds in the following doses (mg/kg): indomethacin (4 and 20); tiaprofenic acid (6 and 30); dicrofenac (20). Mucosal PGI2 was maintained around 50% of the control value in rats given tiaprofenic acid in a dose of 6 mg/kg or dicrofenac in a dose of 4 mg/kg, while indomethacin in a dose of 4 mg/kg markedly reduced mucosal PGI2 to 17% of the control value. In larger doses, tiaprofenic acid and dicrofenac were also significantly less potent in reducing mucosal PGI2 than indomethacin. These results suggest that the difference in ulcerogenicity between indomethacin and the other two compounds was closely related to their potency in decreasing PGI2 in the gastric (fundic) mucosa.     

Retroviral vectors for homologous recombination provide efficient cloning and expression in mammalian cells

Homologous recombination technologies enable high-throughput cloning and the seamless insertion of any DNA fragment into expression vectors. Additionally, retroviral vectors offer a fast and efficient method for transducing and expressing genes in mammalian cells, including lymphocytes. However, homologous recombination cannot be used to insert DNA fragments into retroviral vectors; retroviral vectors contain two homologous regions, the 5′- and 3′-long terminal repeats, between which homologous recombination occurs preferentially. In this study, we have modified a retroviral vector to enable the cloning of DNA fragments through homologous recombination. To this end, we inserted a bacterial selection marker in a region adjacent to the gene insertion site. We used the modified retroviral vector and homologous recombination to clone T-cell receptors (TCRs) from single Epstein Barr virus-specific human T cells in a high-throughput and comprehensive manner and to efficiently evaluate their function by transducing the TCRs into a murine T-cell line through retroviral infection. In conclusion, the modified retroviral vectors, in combination with the homologous recombination method, are powerful tools for the high-throughput cloning of cDNAs and their efficient functional analysis.