immunocytochemical localization of urokinase-type plasminogen activator in lewis lung carcinoma

The invasively growing and metasizing Lewis lung carcinoma consistently contained urokinase-type plasminogen activator (u-PA) enzyme activity. When investigated immunocytochemically with antibodies against u-PA, different parts of individual tumors showed a pronounced heterogeneity in staining intensity. Strong staining was found in areas with invasive growth and degradation of surrounding normal tissue, while other areas were completely devoid of staining. Immunoreactivity occurred both with a perinuclear cytoplasmic localization in tumor cells and associated with apparently extracellular material. SDS PAGE of tumor extracts, under both reducing and nonreducing conditions, followed by immunoblotting, showed only one immunocytochemically stainable band with an electrophoretic mobility corresponding to that of purified proenzyme to u-PA, while no two-chain u-PA was detected. This indicates that the major part of the activator in Lewis lung carcinoma is present as one-chain pro-u-PA.     

Nitrogen and carbon fixation byAnabaena sp. isolated from a rice paddy and grown under P and light limitations

Anabaena sp., isolated from a rice paddy, was investigated for its nitrogen fixation as measured by acetylene reduction activity (ARA) in P-limited continuous and light-limited semi-continuous cultures. Growth rate (μ) under P limitation was a function of cell P content (q _p). Both the photosynthetic capacity (P_max) and photosynthetic efficiency (α) increased with μ when expressed per cell, but not per unit chla. The ARA of steady-state cells under P limitation increased with μ and was linearly related to C-fixation rate. This was apparently a consequence of the control of C-fixation by P limitation. In light-limited cells, steady state ARA, both at the culture light intensity and in the dark, increased asymptotically with μ, but the activity in the dark was only about 51% of that in the light. When the light level of steady-state cells grown at a high in intensity was switched to a low level, ARA decreased exponentially with time. Dark ARA activity also showed a similar decline, but at much lower levels. Thus, ARA depended not only on light history, but also immediate photosynthesis. Steady-state ARA at the ambient intensity or in the dark showed a strong correlation with¹⁴C-fixation rate. ARA of light-limited cells showed the same light-saturation characteristics as their¹⁴C-fixation, with the same initial saturation intensity,I _k. The ratios of P_max to the maximum ARA (ARA_max), and α to the slope of ARA (α_ara) were identical. A comparison of gross to net photosynthesis and N₂ fixation suggested that there was little leakage or excretion of fixed C or N.     

Post-construction avian mortality monitoring at Project West Wind

Abstract

Post-construction avifauna investigations were undertaken at Project West Wind, Meridian Energy Limited's 62-turbine wind farm on the Wellington south coast. These investigations were required in accordance with the resource consent conditions to quantify the level of avian mortalities occurring at the wind farm, particularly in regard to New Zealand falcon (Falco novaeseelandiae), kākā (Nestor meridionalis) and kererū (Hemiphaga novaeseelandiae). This is the first comprehensive study at a New Zealand operating wind farm. The methods included three field components necessary to calculate annual estimates of mortalities across the wind farm site: routine turbine searches; carcass detection trials; and carcass removal trials. Results from years 1 and 2 of a three-year programme are presented. To date, mortalities have been recorded for 17 taxa at 18 of the 24 study turbines. There have been no recorded mortalities of falcon, kākā or kererū. Australasian harrier (Circus approximans) has been the species for which the most mortalities have been recorded. Overall estimated annual mortality rates for years 1 and 2 were calculated to be approximately six and five birds per turbine respectively.     

Genetic architecture of survival and fitness-related traits in two populations of Atlantic salmon

The additive genetic effects of traits can be used to predict evolutionary trajectories, such as responses to selection. Non-additive genetic and maternal environmental effects can also change evolutionary trajectories and influence phenotypes, but these effects have received less attention by researchers. We partitioned the phenotypic variance of survival and fitness-related traits into additive genetic, non-additive genetic and maternal environmental effects using a full-factorial breeding design within two allopatric populations of Atlantic salmon (Salmo salar). Maternal environmental effects were large at early life stages, but decreased during development, with non-additive genetic effects being most significant at later juvenile stages (alevin and fry). Non-additive genetic effects were also, on average, larger than additive genetic effects. The populations, generally, did not differ in the trait values or inferred genetic architecture of the traits. Any differences between the populations for trait values could be explained by maternal environmental effects. We discuss whether the similarities in architectures of these populations is the result of natural selection across a common juvenile environment.     

Therapeutic and pharmacokinetic characterizations of an anti-amyloidogenic bis-styrylbenzene derivative for Alzheimer’s disease treatment

Alzheimer’s disease drug discovery regarding exploration into the molecules and processes has focused on the intrinsic causes of the brain disorder correlated with the accumulation of amyloid-β. An anti-amyloidogenic bis-styrylbenzene derivative, KMS80013, showed excellent oral bioavailability (F = 46.2%), facilitated brain penetration (26%, iv) in mouse and target specific in vivo efficacy in acute AD mouse model attenuating the cognitive deficiency in Y-maze test. Acute toxicity (LD₅₀ >2000 mg/kg) and hERG channel inhibition (14% at 10 μM) results indicated safety of KMS80013.     

Application of a new human cell line,F2N78, in the transient and stable production of recombinant therapeutics

Host cell lines developed by genetic engineering sometimes show instabilities in maintaining their genetically acquired phenotypes. Previously, a hybrid host cell line, designated as hybrid of kidney and B cells (HKB), capable of retaining selected phenotypes originally existing in the parental cells was developed via fusion of 293 cells and HH514‐16 cells. Although HKB did indeed successfully preserve several favorable phenotypes, the expression of Epstein‐Barr virus (EBV) specific nuclear antigen 1 (EBNA1), which should be constitutively expressed for host cells to utilize oriP expression vector in transient production of therapeutic proteins, was observed to be unstable. Here, in an attempt to obtain stable expression of EBNA1, a cell type that contains an integrated EBV genome, rather than HH514‐16 cells, which harbor an episomal EBV genome, was applied for fusion with 293 cells. Fusion of 293 cells with Namalwa cells led to the creation of a new type of hybrid, F2N, which was able to stably express EBNA1 while not producing EBV particles. One of the F2N clones, F2N78, was observed to maintain EBNA1 expression for more than 1 year under serum‐free suspension culture conditions along with human specific glycosyl phenotypes observed previously in HKB. In addition, F2N78 was demonstrated to be an appropriate host cell line for both the transient and stable production of recombinant therapeutics with the features of safety expected of production cell lines for human use. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 432–440, 2013     

Effects of Radix Polygalae on Cognitive Decline and Depression in Estradiol Depletion Mouse Model of Menopause

Postmenopausal syndrome refers to symptoms caused by the gradual decrease in female hormones after mid-40 years. As a target organ of estrogen, decrease in estrogen causes various changes in brain function such as a decrease in choline acetyltransferase and brain-derived neurotrophic factor; thus, postmenopausal women experience cognitive decline and more depressive symptoms than age-matched men. Radix Polygalae has been used for memory boosting and as a mood stabilizer and its components have shown neuroprotective, antidepressant, and stress relief properties. In a mouse model of estrogen depletion induced by 4-vinylcyclohexene diepoxide, Radix Polygalae was orally administered for 3 weeks. In these animals, cognitive and depression-related behaviors and molecular changes related to these behaviors were measured in the prefrontal cortex and hippocampus. Radix Polygalae improved working memory and contextual memory and despair-related behaviors in 4-vinylcyclohexene diepoxide-treated mice without increasing serum estradiol levels in this model. In relation to these behaviors, choline acetyltransferase and brain-derived neurotrophic factor in the prefrontal cortex and hippocampus and bcl-2-associated athanogene expression increased in the hippocampus. These results implicate the possible benefit of Radix Polygalae in use as a supplement of estrogen to prevent conditions such as postmenopausal depression and cognitive decline.     

Use of microorganism-immobilized polyurethane foams to absorb and degrade oil on water surface

Highly oil-absorbent polyurethane foam (PUF) materials were obtained by polymerizing polyether polyol mixture and carbodiimide-modified d-methyl diisocyanate in a weight ratio of 10:2. The foam materials were prepared to contain inorganic nutrients (slow-release fertilizer; SRF) and oil-degrading yeast cells, Yarrowia lipolytica 180, to be applied for removal of oil films on surface waters through absorption and biodegradation after oil spills. PUFs absorbed 7–9 times their own weight of Arabian light crude oil and the oil absorbency appeared to improve as the ratio of surface area to foam weight increased. PUFs showed excellent floatability which was maintained for more than 6 months in sea water, and less than 5% of the absorbed oil was released when the foams were left on water for more than 10 days. For immobilization of yeast cells into PUFs, various immobilization techniques were tested to compare their oil degrading ability and the maintenance thereof. All immobilized cells showed oil degrading abilities as good as those of free cells immediately after the preparation of PUFs, however, the activity of chitin-immobilized cells remained at a high level for the longest period of preservation. The high efficiency of oil absorption and oil degradation by PUF-immobilized yeast cells suggested that PUF-immobilized cells have a high potential as a bioremediation technique for the treatment of oil films on surface waters. Received: 27 September 1999 / Received revision: 6 March 2000 / Accepted: 17 March 2000     

Identification of a cryptic N-terminal signal in Saccharomyces cerevisiae peroxisomal citrate synthase that functions in both peroxisomal and mitochondrial targeting

Saccharomyces cerevisiae has three distinct citrate synthases, two located in mitochondria (mature Cit1p and Cit3p) and one in peroxisomes (mature Cit2p). While the precursor of the major mitochondrial enzyme, Cit1p, has a signal for mitochondrial targeting at its N-terminus (MTS), Cit2p has one for peroxisomal targeting (PTS1) at its C-terminus. We have previously shown that the N-terminal segment of Cit2p is removed during import into peroxisomes [Lee, H.S. et al. (1994) Kor. J. Microbiol. 32, 558-564], which implied the presence of an additional N-terminal sorting signal. To analyze the function of the N-terminal region of Cit2p in protein trafficking, we constructed the N-terminal domain-swapped versions of Cit1p and Cit2p. Both fusions, Cit1::Cit2 and Cit2::Cit1, complemented the glutamate auxotrophy caused by the double-disruption of the CIT1 and CIT2 genes. In addition, part of the Cit2::Cit1 fusion protein, as well as Cit1::Cit2, was shown to be transported into both mitochondria and peroxisomes. The subcellular localization of the recombinant fusion proteins containing various N-terminal segments of Cit2p fused to a mutant version of green fluorescent protein (GFP2) was also examined. As a result, we found that the 20-amino acid N-terminal segment of Cit2p contains a cryptic cleavable targeting signal for both peroxisomes and mitochondria. In addition, we show that the peroxisomal import process mediated by the N-terminal segment of Cit2p was not affected by the disruption of either PEX5 (encoding PTS1 receptor) or PEX7 (encoding PTS2 receptor).