Intraocular gutless adenoviral-vectored VEGF stimulates anterior segment but not retinal neovascularization

Vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) have been implicated as important stimulatory factors for retinal neovascularization. In this study, we used intraocular gene transfer with gutless adenoviral (AGV) vectors to determine the effect of increased intraocular expression of VEGF, IGF-1, or sphingosine kinase (SPK), which produces sphingosine-1-phosphate, another angiogenic factor. Retinal neovascularization did not occur from intravitreous AGV-vectored VEGF, IGF-1, SPK, or combined VEGF and IGF-1, except occasionally adjacent to the retinal penetration site from the injection. However, corneal and iris neovascularization occurred after 2 weeks in all eyes injected with AGV.VEGF, but not those injected with only AGV.IGF-1 or AGV.SPK. These data suggest that the superficial capillary bed of the retina is relatively insensitive to VEGF, IGF-1, or SPK in adult mice, except when combined with retinal trauma. However, AGV-vectored VEGF is sufficient to consistently cause severe corneal and iris neovascularization. This provides a model for anterior segment neovascularization, which unlike previous models is relatively inexpensive and is not plagued by spontaneous regression, and therefore, may be useful for identification of new treatments.     

Morphological differences between guinea pig aortic and venous endothelial cells in situ

Endothelial cells (ECs) respond to fluid shear stress. They reveal shear stress related morphological changes in both their cell shape and cytoskeletal organization. Little is known about the cytoskeletal organization of ECs in situ. We studied, together with the living ultrasound high resolution imaging system, the distribution of stress fibers (SFs), certain focal adhesion (FA) and signal transduction associated proteins in guinea pig aortic and venous ECs. Although SFs present in the basal portion of venous ECs ran along the direction of the blood flow, their size was smaller and their number was fewer than those of aortic ECs. Venous ECs were elongated to the direction of flow than in aortic ECs exposed over normal shear stress (SS). Since fluid SS in the vein is low, a sustained and uni-directional low SS over a long period might thus cause these structural features observed in venous ECs.     

Auxin Produced by the Indole-3-Pyruvic Acid Pathway Regulates Development and Gemmae Dormancy in the Liverwort Marchantia polymorpha

The plant hormone auxin (indole-3-acetic acid [IAA]) has previously been suggested to regulate diverse forms of dormancy in both seed plants and liverworts. Here, we use loss- and gain-of-function alleles for auxin synthesis- and signaling-related genes, as well as pharmacological approaches, to study how auxin regulates development and dormancy in the gametophyte generation of the liverwort Marchantia polymorpha. We found that M. polymorpha possess the smallest known toolkit for the indole-3-pyruvic acid (IPyA) pathway in any land plant and that this auxin synthesis pathway mainly is active in meristematic regions of the thallus. Previously a Trp-independent auxin synthesis pathway has been suggested to produce a majority of IAA in bryophytes. Our results indicate that the Trp-dependent IPyA pathway produces IAA that is essential for proper development of the gametophyte thallus of M. polymorpha. Furthermore, we show that dormancy of gemmae is positively regulated by auxin synthesized by the IPyA pathway in the apex of the thallus. Our results indicate that auxin synthesis, transport, and signaling, in addition to its role in growth and development, have a critical role in regulation of gemmae dormancy in M. polymorpha.     

A splice variant of the TCR zeta mRNA lacking exon 7 leads to the down-regulation of TCR zeta, the TCR/CD3 complex, and IL-2 production in systemic lupus erythematosus T cells

The reduction or absence of TCR zeta-chain (zeta) expression in patients with systemic lupus erythematosus (SLE) is thought to be a factor in the pathogenesis of SLE. We previously reported a splice variant of zeta mRNA that lacks the 36-bp exon 7 (zeta mRNA/exon 7(-)) and is accompanied by the down-regulation of zeta protein in T cells from SLE patients. In this study, we show that EX7- mutants (MA5.8 cells deficient in zeta protein that have been transfected with zeta mRNA/exon 7(-)) exhibit a reduction in the expression of TCR/CD3 complex and zeta protein on their cell surface as well as a reduction in the production of IL-2 after stimulation with anti-CD3 Ab, compared with that in wild-type (WT) mutants (MA5.8 cells transfected with the WT zeta mRNA). Furthermore, real-time PCR analyses demonstrated that zeta mRNA/exon 7(-) in EX7- mutants was easily degraded compared with zeta mRNA by the WT mutants. Pulse-chase experiment showed zeta protein produced by this EX7- mutants was more rapidly decreased compared with the WT mutants. Thus, the lower stability of zeta mRNA/exon 7(-) might also be responsible for the reduced expression of the TCR/CD3 complex, including zeta protein, in SLE T cells.     

Quantitative determination of tulobuterol and its metabolites in human urine by mass fragmentography

A method is described for the simple and simultaneous determination of tulobuterol and its metabolites in human urine by gas chromatography-mass spectrometry. Quantification was achieved by single-ion monitoring at m/e 86 derived from trimethylsilyl-tulobuterol and its metabolites using a column packed with a mixed phase, 2% OV-1–2% QF-1 (1 : 1, w/w). The detection limits were estimated to be 2 ng/ml in urine for tulobuterol and 5 ng/ml for metabolites, respectively.     

Reproductive biology and genetic population structure of two alien Lolium species inhabiting the sandy coasts of Japan

Understanding the characteristics of alien species is a prerequisite for any biological study or anti-invasion management strategy. Lolium rigidum (Poaceae) is an alien species that has become naturalized on the sandy coasts of Japan; however, it exhibits extensive morphological variation, leading to speculation that several taxa of Lolium have become naturalized. Here, we compared the morphology, reproductive biology and genetic structure of this Lolium species by growing individuals from different locations in the same environment to clarify whether the observed morphological variation is genetically based or is caused by intraspecific variation as a result of environmental differences. Principle component analyses of 11 morphological traits separated the study species into two types. Bagging experiments showed that one type exhibited an outcrossing life history, whereas the other type selfed exclusively. Nuclear DNA microsatellite analyses supported this distinction between these morphological types, with no intermediate individuals being found. The outcrossing type exhibited high genetic diversity, whereas the selfing type exhibited almost no polymorphism, reflecting the differences in their breeding systems. Moreover, both types differed from the economically important outcrossing species Lolium multiflorum and Lolium perenne. These two types of Lolium are expected to have different introduction histories and invasive potential on the coasts of Japan. In conclusion, it is important to distinguish between these types through additional biological studies to design effective control measures.     

LARGE SCALE BIOSYNTHESIS OF GANGLIOSIDE ANALOGUES BY RERF-LC-AI CELLS CULTURED IN HYPERFlask

The efficient production of ganglioside analogues was accomplished using RERF-LC-AI cells cultured in HYPERFlask (High Yield PERformance Flask). Eight kinds of ganglioside analogues (GM3, GM2, sialylparagloboside, GD3, di-sialylated lacto-N-tetraose, and another three kinds of analogues with intricate structures) were synthesized by the saccharide primer method using lung squamous-cell carcinoma line RERF-LC-AI and 12-azidododecyl β-lactoside primer. The yield for each analogue obtained using HYPERFlask was higher than yields obtained from 100-mm dishes.     

Potential use of the astaxanthin-producing microalga, <Emphasis Type=Italic>Monoraphidium</Emphasis> sp. GK12, as a functional aquafeed for prawns

We determined the nutritional profile of Monoraphidium sp. GK12, a newly isolated astaxanthin (AXA)-producing microalga, and investigated its potential use as a functional aquafeed by evaluating its effect on prawn pigmentation. GK12 contained high levels of pantothenic acid. The β-carotene content of GK12 was higher than that of Haematococcus, a well-studied AXA producer, and was similar to that of Spirulina. GK12 also had a high content of unsaturated fatty acids, of which linolenic acid (C18:3 n−3) was the most plentiful. A GK12-containing feed resulted in significant pigmentation of the prawns, comparable to that of prawns fed on synthetic AXA or Haematococcus. A GK12-containing feed also increased the survival rate of the prawns. Therefore, in addition to improving cultivation methods for Haematococcus, further research is needed into the use of GK12 as an alternative AXA source and as an ingredient of functional aquafeed for farmed fish.     

Lysosomal glycogen accumulation in rat liver and its in vivo kinetics after a single intraperitoneal injection of acarbose, an alpha-glucosidase inhibitor

A single intraperitoneal injection of acarbose (400 mg/kg) into rats caused lysosomal accumulation of glycogen in the liver, mimicking the cytological characteristics of human glycogen storage disease type II (Pompe's disease). The animal model is therefore useful for studying the pathogenesis of the disease. In the present study, we applied this model to examine the lysosomal hydrolytic pathway of glycogen in vivo. To quantify the lysosomal glycogen, the lysosome-rich fraction was rapidly prepared from liver homogenate by agglutination in the presence of Ca2+. Then the fraction was treated with alpha-amylase in isotonic medium to remove cytosolic glycogen, followed by transfer to hypotonic conditions in the presence of Triton X-100 to destroy total glycogen. The amount of lysosomal glycogen was calculated from the difference between the glycogen levels measured before and after the treatment under hypotonic conditions, and then it was corrected based on measurements of the intactness (%) of lysosomes and the recovery (%) of the lysosomal marker enzyme (beta NAGase). We observed no measurable lysosomal glycogen in normal liver by this method, and this was confirmed by electron microscopy. After administration of acarbose, the lysosomal glycogen level increased to 2.5 mg/g liver within 2 days, and then decreased gradually at a rate of 0.4 mg/day/g. The accumulation of glycogen in the lysosomes at an initial velocity of 1.5 mg/day/g liver may be considered as the amount of glycogen that would normally be degraded by acid alpha-glucosidase. Therefore, assuming that the liver breaks down about 40 mg glycogen/day/g, we estimated that about 3% of the glycogen would be hydrolyzed by the lysosomal pathway.