Inhibition of established subcutaneous and metastatic murine tumors by intramuscular electroporation of the interleukin-12 gene

In vivo electroporation (EP) of the murine interleukin-12 (IL-12) gene in an expression plasmid (pIL-12) was evaluated for antitumor activity. EP transfer of pIL-12 into mouse quadriceps muscles elicited significant levels of serum IL-12 and interferon-gamma. Intramuscular EP of pIL-12 resulted in complete regression or substantial inhibition of 38C13 B-cell lymphoma, whereas pIL-12 delivered by gene gun or intramuscular injection without EP showed little therapeutic effect. Impressive antitumor activity by intramuscular EP was also demonstrated in animals with advanced malignant disease. At day 14 after 38C13 tumor inoculation, all animals were found to carry large tumors and to have metastases; without treatment, most died within a week. A single intramuscular EP of pIL-12 resulted in regression of 50% of large subcutaneous tumors and significantly prolonged the lifespan of these animals. Moreover, animals that were previously cured of 38C13 tumors by in vivo EP treatment significantly suppressed tumor growth when challenged 60 days later. In vivo EP of the IL-12 gene was also effective in suppressing subcutaneous and lung metastatic tumors of CT-26 colon adenocarcinoma and B16F1 melanoma cells. Together, these results show that intramuscular electrotransfer of the IL-12 gene may represent a simple and effective strategy for cancer treatment.     

Snake inhibitors of phospholipase A2 enzymes

Phospholipase A₂ (PLA₂) enzymes consist of a large family of proteins which share the same enzymatic function and display considerable sequence homology. These enzymes have been identified and characterised in mammalian tissue and snake venoms. Numerous physiological functions have been attributed to mammalian PLA₂s and they are nontoxic. In comparison, venom PLA₂s are toxic and induce a variety of pharmacological effects that are probably mediated via membrane receptors. Snake PLA₂ inhibitors (PLIα), with a similar structure to the M-type receptor, have been identified as soluble complexes in the serum of viperinae and crotalinae snakes. These inhibitors showed selective binding to crotalid group II PLA₂s and appeared to be restricted to the serum of this snake family. Analysis of PLA₂ binding to recombinant fragments of PLIα indicated that the CRD region was most likely responsible for enzyme inhibition. A second type of inhibitor, PLIβ, has been identified in serum from one viperid snake and consists of a leucine-rich structure. The third type of inhibitor, PLIγ, was found in the serum of five snake families and contains a pattern of cysteine residues that define a three-finger structure. PLIγ inhibitors isolated from the serum of Elapidae, Hydrophidae, Boidae and Colubridae families were able to inhibit a broad range of enzymes including the nontoxic mammalian group IB and IIA PLA₂s, and bee venom group III PLA₂. However, differences in the binding affinities indicated specificity for particular PLA₂s. A different representation has emerged for crotalid and viperid snakes. Their PLIγs did not inhibit bee venom group III, mammalian group IB and IIA enzymes. Furthermore, inhibition data for the γ-type inhibitor from Crotalus durissus terrificus (CICS) showed that this inhibitor was specific for viperid β-neurotoxins and did not inhibit β-neurotoxins from elapids [1]. Further studies are required to determine if this phenomenon is true for all γ-type inhibitors from Crotalidae snakes. The relative distribution of these inhibitors, their specificities and the structural features involved in binding are discussed in this review.     

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.     

Yeast diversity in hypersaline habitats

Thus far it has been considered that hypersaline natural brines which are subjected to extreme solar heating, do not contain non-melanized yeast populations. Nevertheless we have isolated yeasts in eight different salterns worldwide, as well as from the Dead Sea, Enriquillo Lake (Dominican Republic) and the Great Salt Lake (Utah). Among the isolates obtained from hypersaline waters, Pichia guilliermondii, Debaryomyces hansenii, Yarrowia lipolytica and Candida parapsilosis are known contaminants of low water activity food, whereas Rhodosporidium sphaerocarpum, R. babjevae, Rhodotorula laryngis, Trichosporon mucoides, and a new species resembling C. glabrata were not known for their halotolerance and were identified for the first time in hypersaline habitats. Moreover, the ascomycetous yeast Metschnikowia bicuspidata, known to be a parasite of the brine shrimp, was isolated as a free-living form from the Great Salt Lake brine. In water rich in magnesium chloride (bitterns) from the La Trinitat salterns (Spain), two new species provisionally named C. atmosphaerica - like and P. philogaea - like were discovered.     

Cytogenetic and molecular characterization of the Abies alba genome and its relationship with other members of the Pinaceae

Genome size, karyotype structure, heterochromatin distribution, position and number of ribosomal genes, as well as the ITS2 sequence of the internal transcribed spacer (ITS) were analysed in silver fir (Abies alba Mill.). The analysis also included characterization of the Arabidopsis-type of telomeric repeats in silver fir and in related species. The results were compared with results from other species of the Pinaceae, to evaluate phylogeny and chromosomal and molecular evolution in the Pinaceae. Integrated chromosomal data provided insights into chromosome and karyotype evolution in the Pinaceae. The evolutionary trend for GC-rich heterochromatic blocks seems to involve loss of blocks that are not associated with rDNA. Similarly, numerous large blocks of interstitial plant telomeric repeats that are typical for all analysed species of the genus Pinus were not observed in the evolutionarily younger genera, such as Abies, Picea and Larix. On the contrary, the majority of telomeric sequences in these three genera appeared confined to the chromosome ends. We confirmed the current position of Abies and Tsuga in subfamily Abietoideae and the position of Pinus in the subfamily Pinoideae based on ITS2 sequences. Pseudotsuga is placed together with Larix into the subfamily Laricoideae. We conclude that the current position of the genus Picea in the subfamily Abietoideae should be reconsidered and, possibly, the genus Picea should be reclassified as a separate subfamily, Piceoideae, as recently proposed.     

Human embryonic stem cells and metastatic colorectal cancer cells shared the common endogenous human microRNA‐26b

The increase in proliferation and the lack of differentiation of cancer cells resemble what occur in the embryonic stem cells during physiological process of embryogenesis. There are also striking similarities in the behaviour between the invasive placental cells and invasive cancer cells. In the present study, microarrays were used to analyse the global expression of microRNAs in a human embryonic stem cell line (i.e. HUES‐17) and four colorectal cancer (CRC) cell lines (i.e. LoVo, SW480, HT29 and Caco‐2) with different metastatic potentialities. Only the expression of miR‐26b was significant decreased in HUES‐17s and LoVo cells, compared with other three cell lines (P < 0.01). The quantitative real‐time PCR analysis confirmed the results of the microarray analysis. Overexpression of miR‐26b expression by miR‐26 mimics transfection and led to the significant suppression of the cell growth and the induction of apoptosis in LoVo cells in vitro, and the inhibition of tumour growth in vivo. Moreover, the potential targets of miR‐26b was predicted by using bioinformatics, and then the predicted target genes were further validated by comparing gene expression profiles between LoVo and NCM460 cell lines. Four genes (TAF12, PTP4A1, CHFR and ALS2CR2) with intersection were found to be the targets of miR‐26b. MetaCore network analysis further showed that the regulatory pathways of miR‐26b were significantly associated with the invasiveness and metastasis of CRC cells. These data suggest that miR‐26b might serve as a novel prognostic factor and a potential therapeutic target for CRC.     

Feedback mechanisms regulate retinoic acid production and degradation in the zebrafish embryo

Retinoic acid (RA) signaling in vertebrate embryos occurs in a distinct physical and temporal pattern. Regulating this spatial distribution is crucial to the development of the embryo, as RA in excess or in inappropriate tissues is teratogenic. In order to understand how RA availability is determined in zebrafish we have investigated the expression of cyp26a1, an enzyme that inactivates RA, and its relationship to raldh2, one of the enzymes that produce RA from retinal. cyp26a1 expression follows three phases: in presumptive anterior neurectoderm and in a circumblastoporal ring during gastrulation, in the tailbud throughout somitogenesis, and in multiple specific tissue types beginning at mid-somitogenesis and continuing through 48 h postfertilization (hpf). This expression was either adjacent or opposite to those tissues expressing raldh2. We then investigated how RA production might regulate these relationships. Endogenous RA produced by raldhs did not play a role in setting cyp26a1 expression in most tissues. However, exogenous RA regulates expression of both enzymes. cyp26a1 is up regulated in the embryo in a time, concentration, and tissue-dependent manner. Conversely, raldh2 expression is reduced with RA treatment. Tests of the raldh2 promoter in cell transfections proved that RA directly represses its activity. These data demonstrate that the feedback mechanisms regulating production and degradation of RA must be considered in any experiments altering levels of RA in the developing vertebrate embryo.     

Inhibitory effect of genistein on mouse colon cancer MC-26 cells involved TGF-beta1/Smad pathway

TGF-beta1/signaling has been shown to be associated with proapoptotic and antimitotic activities in epithelial tissues. Genistein, a major component of soybean isoflavone, has multiple functions resulting in anticancer proliferation. We herein showed that genistein dose-dependently increased TGF-beta1 mRNA expression in mouse colon cancer MC-26 cells. A mouse monoclonal anti-TGF-beta1 neutralizing antibody partially, but not completely, blocked the growth inhibition by genistein. By using adenoviral vector, we demonstrated that Smad7 overexpression attenuated genistein-induced growth inhibition and apoptosis as determined by MTT and apoptosis ELISA. Smad7 overexpression also inhibited upregulation of p21 and caspase-3 activity by geinistein. To further confirm inhibitory effect of genistein in MC-26 cells require TGF-beta1/Smad signaling, we employed Western blot and electrophoretic mobility shift assay to detect formation of Smad-DNA complexes and phosphorylation of Smad2 and Smad3, respectively. Data revealed that genistein induced an evident formation of Smad-DNA complexes and phosphorylation of Smad2 and Smad3, indicating increased TGF-beta1 signaling. Taken together, these findings first provided insights into possible molecular mechanisms of growth inhibition by genistein that required Smad signaling, which could aid in its evaluation for colon tumor prevention.     

解脂亚罗酵母菌体外降甘油三酯降胆固醇效果研究

筛选出高效降三酰甘油降胆固醇菌株,为新型药物、食品的制备提供优良菌株。从样品中筛选分离出酵母菌株,并测定菌株降三酰甘油能力,采用磷硫铁比色法测定菌株降胆固醇能力,通过耐酸、耐胆盐及毒性实验研究菌株各项性能,通过生理生化及26S rDNA法对菌株进行鉴定。结果显示,试验筛选出1株高效降脂降胆固醇酵母菌EAM-ZT003T,降三酰甘油能力高达80.2%,菌株具有很好的耐酸、耐胆盐能力,为期42 d的小鼠毒性试验发现,小鼠未出现异常体征,未发现异常死亡,且解剖观察各器官一切正常,菌株经26S rDNA鉴定为解脂亚罗酵母菌(Yarrowia lipolytica)。     

CYP26A1 and CYP26C1 cooperatively regulate anterior-posterior patterning of the developing brain and the production of migratory cranial neural crest cells in the mouse

The appropriate regulation of retinoic acid signaling is indispensable for patterning of the vertebrate central nervous system along the anteroposterior (A-P) axis. Although both CYP26A1 and CYP26C1, retinoic acid-degrading enzymes that are expressed at the anterior end of the gastrulating mouse embryo, have been thought to play an important role in central nervous system patterning, the detailed mechanism of their contribution has remained largely unknown. We have now analyzed CYP26A1 and CYP26C1 function by generating knockout mice. Loss of CYP26C1 did not appear to affect embryonic development, suggesting that CYP26A1 and CYP26C1 are functionally redundant. In contrast, mice lacking both CYP26A1 and CYP26C1 were found to manifest a pronounced anterior truncation of the brain associated with A-P patterning defects that reflect expansion of posterior identity at the expense of anterior identity. Furthermore, Cyp26a1-/-Cyp26c1-/- mice fail to produce migratory cranial neural crest cells in the forebrain and midbrain. These observations, together with a reevaluation of Cyp26a1 mutant mice, suggest that the activity of CYP26A1 and CYP26C1 is required for correct A-P patterning and production of migratory cranial neural crest cells in the developing mammalian brain.