Role of eNOS in neovascularization: NO for endothelial progenitor cells

Nitric oxide (NO) is a gaseous molecule with an astonishingly wide range of physiological and pathophysiological activities, including the regulation of vessel tone and angiogenesis in wound healing, inflammation, ischaemic cardiovascular diseases and malignant diseases. Recent data have revealed the predominant role of endothelial nitric oxide synthase (eNOS), an endothelial-cell-specific isoform of NO producing enzyme, in both angiogenesis (the development of new blood vessels derived from existing vessels) and vasculogenesis (blood vessel formation de novo from progenitor cells). In addition, successes in gene therapy, together with the recent development of an eNOS-specific inhibitor, suggest that the modulation of eNOS might be a potent new strategy for the control of pathological neovascularization.     

Reaction characteristics of an immobilized yeast producing ethanol

The reaction behavior of Saccharomyces formaosensis imobilized by polyacrylamide gel is presented. Two types of the immobilized yeast are studied, i. e. the immobilized resting yeast and the immobilized growing yeast. For both of the yeast, reaction retes are expressed by the Michaelis-Menten type equation with a linear ethanol inhibition factor. The Michaelis constants aere close each other, but considerably larger that of native S. cerevisiae. Distribution of the growing yeast cell inside the carrier gel is presented. It is found that the cell density is somewhat higher near the surface of the carrier.     

Stepwise loss of metabolism of ε-aminocaproic acid cyclic dimer in Alcaligenes species D-2

Summary A bacterium which is able to utilize the cyclic dimer of -aminocaproic acid (ACA) as a sole source of carbon and nitrogen has been isolated, classified as a member of Alcaligenes and tentatively named D-2. The initial step of the ACA cyclic dimer metabolism in D-2 may be composed of the following three reactions, which are catalyzed by specific enzymes: opening of the ACA cyclic dimer, splitting of the ACA linear dimer and transamination of ACA. By treatment with mitomycin C or ethidium bromide, 2–3% of the D-2 cells lost both ACA cyclic dimer-opening and ACA linear dimer-splitting activities. Slow growth of colonies of this variant strain on ACA agar medium kept at 12±3° C for 4 weeks resulted in the production of a new variant which had lost the ACA transaminase activity as well as the hydrolysis activities. When the parent strain (D-2) was grown slowly on ACA cyclic dimer agar medium in the same way, the ACA transaminase activity alone was lost by about 30% of the colonies. All the variants have been stable during 6 months of culture by successive transfer on agar media. These facts suggest that both the ACA cyclic dimer-opening enzyme and the ACA linear dimer-splitting enzyme are encoded by the same plasmid whereas the ACA transaminase is encoded by a second plasmid.     

Embedding a grammatical description in deterministic chaos: an experiment in recurrent neural learning

We consider the modeling process of a biological agent by combining the concepts of neuroinformatics and deterministic chaos. We assume that an agent observes a target process as a stochastic symbolic process, which is restricted by grammatical constraints. Our main hypothesis is that an agent would learn the target model by reconstructing an equivalent quasi-stochastic process on its deterministic neural dynamics. We employed a recurrent neural network (RNN), which is regarded as an adjustable deterministic dynamical system. Then, we conducted an experiment to observe how the RNN learns to reconstruct the target process, represented by a stochastic finite state machine in the simulation. The result revealed the capability of the RNN to evolve, by means of learning, toward chaos, which is able to mimic a target's stochastic process. We precisely analyzed the evolutionary process as well as the internal representation of the neural dynamics obtained. This analysis enabled us to clarify an interesting mechanism of the self-organization of chaos by means of neural learning, and also showed how grammar can be embedded in the evolved deterministic chaos.     

RGD–cyclam conjugate: Synthesis and potential application for positron emission tomography

Cyclam and DOTA-containing positron emission tomography radiotracers were prepared by using a modular chemical strategy based on peptide synthesis and chemoselective ligations. These molecules encompass two functional domains, one a tumour ‘homing’ domain and the other a chelating ligand for copper allowing nuclear imaging of tumours.     

Histopathologic findings and establishment of novel tumor lines from spontaneous tumors in FVB/N mice

The inbred FVB mouse strain is used extensively in cancer research. Transgenic mice with an FVB/N background in which the expression of green fluorescent protein is under the control of various promoters have been used widely for the last decade. However, little is known about the incidence and characteristics of spontaneous tumors in these mice. In addition, only a few tumor lines have been established for use in this particular mouse strain. Our aim was to initiate a database of spontaneous tumors in our retired FVB/N breeders, analyze the histopathologic characteristics of these tumors, and establish novel tumor lines in vivo and in vitro. A total of 234 (40 male, 194 female) breeder mice were observed during their natural lifespans. The incidence of spontaneous tumors was 45.0% in male mice and 52.8% in female mice. All tumors in male mice were lung alveolar-bronchiolar (AB) neoplasms, except for 1 testis interstitial cell tumor. In female mice, histopathologic examination revealed 48 lung AB tumors, 27 mammary gland tumors, 13 ovarian tumors, and 14 other tumors. Several of these spontaneous tumors have been transplanted into FVB/N mice. One mammary adenocarcinoma (MCaP0008) and 1 lung AB carcinoma (LAP0297) were successfully transplanted subcutaneously and passaged serially in vivo. Subsequently, we established cell lines from both tumors, which were maintained in monolayer in vitro. Both of the grafted tumors and cell lines are tumorigenic in VEGF(P)-GFP/FVB and Tie2(P)-GFP/FVB mice. Establishment of these novel tumor lines will benefit both in vivo and in vitro studies on the pathophysiology of cancer in this relatively new but widely used mouse strain.     

Effects of adipokinetic hormone and its related peptide on maintaining hemolymph carbohydrate and lipid levels in the two-spotted cricket,Gryllus bimaculatus

Adipokinetic hormone (AKH) regulates energy homeostasis in insects by mobilizing lipid and carbohydrate from the fat body. Here, using RNA sequencing data, we identified cDNAs encoding AKH (GbAKH) and its highly homologous hormone AKH/corazonin-related peptide (GbACP) in the corpora cardiaca of the two-spotted cricket, Gryllus bimaculatus. RT-PCR revealed that GbAKH and GbACP are predominantly expressed in the corpora cardiaca and corpora allata, respectively. Phylogenetic analysis confirmed that the identified GbAKH and GbACP belong to the clades containing other AKHs and ACPs, respectively. Injection of synthetic GbAKH and GbACP elevated hemolymph carbohydrate and lipid levels and reduced food intake significantly. In contrast, knockdown of GbAKH and GbACP by RNA interference increased the food intake, although hemolymph lipid level was not altered. Collectively, this study provides evidence that ACP regulates hemolymph carbohydrate and lipid levels in cricket, possibly collaborative contribution with AKH to the maintenance of energy homeostasis.     

Acetaldehyde-Derived Advanced Glycation End-Products Promote Alcoholic Liver Disease

Background

Chronic ingestion of ethanol increases acetaldehyde and leads to the production of acetaldehyde-derived advanced glycation end-products (AA-AGE). We evaluated the toxicity of AA-AGE on hepatocytes and studied the role of AA-AGE in the pathogenesis of alcoholic liver disease (ALD).

Methods

Rat hepatocyte cultures were treated with N-ethyllysine (NEL) or AA-AGE and the cell viability was evaluated using MTT assay. Male Wistar rats were fed with liquid diet containing 5% ethanol for 8 weeks following normal diet for another 12 weeks. A group of animals was sacrificed at 4th, 6th, and 8th week and the remaining animals at 12th, 14th, 16th, 18th, and 20th week. The liver sections were stained for AA-AGE and 4-hydroxy-2-nonenal (4-HNE). Liver biopsy obtained from ALD patients was also stained for AA-AGE and 4-HNE.

Results

Hepatocyte viability was significantly reduced in cultures treated with AA-AGE compared to NEL treated or control cultures. Severe fatty degeneration was observed during chronic administration of ethanol increasing from 4–8 weeks. The staining of AA-AGE and 4-HNE was correlated with the degree of ALD in both rat and human. In rats, hepatic fatty degeneration was completely disappeared and the staining for both AA-AGE and 4-HNE returned to normal at 12th week of abstinence. Staining for AA-AGE and 4-HNE was completely absent in normal human liver.

Conclusions

The data demonstrated that AA-AGE is toxic to hepatocytes, but not NEL. Chronic ethanol ingestion produces AA-AGE and reactive oxygen species that contribute to the pathogenesis of ALD. Abstinence of alcohol results in complete disappearance of both AA-AGE and 4-HNE along with fatty degeneration suggesting that AA-AGE plays a significant role in the pathogenesis of ALD.     

Green fluorescent protein (GFP)-expressing tumor model derived from a spontaneous osteosarcoma in a vascular endothelial growth factor (VEGF)-GFP transgenic mouse

Vascular endothelial growth factor (VEGF) mediates tumor angiogenesis, growth, and metastasis. Murine models of metastatic tumors in which green fluorescent protein (GFP) expression is driven by the VEGF promoter can be imaged both intravitally and externally and thus offer many possibilities for real-time studies of tumor angiogenesis, metastasis, and treatment in vivo. In our defined-flora animal facility, an 11-month-old female transgenic mouse with a C3H background (VEGF(P)-GFP/C3H) developed a spontaneous tumor that expressed GFP under the control of VEGF. Necropsy and histopathologic examination revealed an osteosarcoma with lung metastases. Fresh tumor fragments were transplanted successfully into other VEGF(P)-GFP/C3H transgenic mice. During the first five generations, the tumor "take rate" was 100% (25 of 25 animals), with a latent period of 8 days and an average tumor volume of 1500 mm3 at 36 days. Transplanted tumors have maintained their original histopathologic characteristics and metastatic behavior. In addition, the tumor grows in wild-type C3H mice with an 83% take rate (10 of 12 animals) and as monolayer cells in vitro. GFP was expressed strongly in tumor tissue, lung metastatic foci, and cultured tumor cells. Real-time growth of tumors grown in dorsal skin chambers in C3H mice could be visualized using GFP fluorescence. In addition, GFP fluorescence of metastatic lesions in lungs of C3H mice was clearly visible by multiphoton laser scanning microscopy. This in vitro and in vivo transplantable and metastatic osteosarcoma (Os-P0107) is an attractive model for further study of tumor pathophysiology and treatment efficiency affecting VEGF expression.