In vitro remodeling of tumor vascular endothelial cells using conditioned medium from various tumor cells and their sensitivity to TNF-alpha

Prevention of tumor-associated blood vessel formation (angiogenesis) is a potentially powerful strategy to treat cancer. We found that tumor vascular endothelial cells were rearranged in vitro with conditioned culture medium derived from tumor cells and compared the sensitivity to the effects of TNF-alpha between normal and tumor endothelial cells. Incubation with tumor (Meth-A, Colon26)-derived conditioned medium showed that no effect was observed on cell growth. Tumor cells (Meth-A, Colon26, and B16BL6) only showed no sensitivity to TNF-alpha. Normal and control endothelial cells in culture showed little cytotoxicity in response to TNF-alpha treatment, but marked cytotoxicity of TNF-alpha was observed in endothelial cells cultured with tumor-derived conditioned medium. Sensitivity to TNF-alpha was different depending on the type of tumor from which the conditioned medium was derived. This difference in sensitivity was assumed to be due to the in vivo sensitivity to TNF-alpha. The results of this study suggested that the sensitivity of tumors to TNF-alpha is controlled by the sensitivity of tumor vasculature.     

Environmental factors which influence the sink of silica in the limnetic system of the large monomictic Lake Biwa and its watershed in Japan

Dissolved silica (DSi) and its associated biological and physicochemical factors were measured in Lake Biwa, Japan and its watershed from 2002 to 2003 in order to clarify seasonal variations in the magnitude of the sink of silica and the factors that influence it within the limnetic system. Consequently, it is concluded that Lake Biwa is a noticeable body of water where a massive sink of silica is caused. Calculated silica sedimentation in Lake Biwa was 2.0 × 10⁷ kg Si year⁻¹ (7.1 × 10⁸ mol Si year⁻¹) which is equivalent to about 80% of the annual inflow discharge of DSi to Lake Biwa. The magnitude of the sink varies seasonally by increasing in the winter holomictic stirring period, since it is greatly affected by the species composition of phytoplankton, the load of phosphorus and the condition of stratification. It seems reasonable to suppose that the DSi in Lake Biwa is removed mainly by biological processes, i.e., the assimilation of DSi by large centric diatoms and its accumulation in their frustules. Such silica sinks occur naturally in deeper stagnant waters, providing extended water residence time and supplying a certain amount of nutrients. These findings indicate that an increase in nutrient loads and abundance of stagnant water due to the construction of large dams lead to an expansion in the magnitude of the silica sink in a limnetic system.     

Nitrosonifedipine Ameliorates the Progression of Type 2 Diabetic Nephropathy by Exerting Antioxidative Effects

Diabetic nephropathy (DN) is the major cause of end-stage renal failure. Oxidative stress is implicated in the pathogenesis of DN. Nitrosonifedipine (NO-NIF) is a weak calcium channel blocker that is converted from nifedipine under light exposure. Recently, we reported that NO-NIF has potential as a novel antioxidant with radical scavenging abilities and has the capacity to treat vascular dysfunction by exerting an endothelial protective effect. In the present study, we extended these findings by evaluating the efficacy of NO-NIF against DN and by clarifying the mechanisms of its antioxidative effect. In a model of type 2 DN (established in KKAy mice), NO-NIF administration reduced albuminuria and proteinuria as well as glomerular expansion without affecting glucose metabolism or systolic blood pressure. NO-NIF also suppressed renal and systemic oxidative stress and decreased the expression of intercellular adhesion molecule (ICAM)-1, a marker of endothelial cell injury, in the glomeruli of the KKAy mice. Similarly, NO-NIF reduced albuminuria, oxidative stress, and ICAM-1 expression in endothelial nitric oxide synthase (eNOS) knockout mice. Moreover, NO-NIF suppressed urinary angiotensinogen (AGT) excretion and intrarenal AGT protein expression in proximal tubular cells in the KKAy mice. On the other hand, hyperglycemia-induced mitochondrial superoxide production was not attenuated by NO-NIF in cultured endothelial cells. These findings suggest that NO-NIF prevents the progression of type 2 DN associated with endothelial dysfunction through selective antioxidative effects.     

The structure of the second cytosolic loop of the yeast mitochondrial ADP/ATP carrier AAC2 is dependent on the conformational state

To detect structural changes in the second cytosolic loop of the mitochondrial ADP/ATP carrier of Saccharomyces cerevisiae AAC2, we prepared 20 single cysteine mutants by replacing each amino acid in the S213 to L232 region. All single cysteine mutants were fully functional, because they could restore growth on glycerol of a yeast strain lacking functional ADP/ATP carriers. First, these single-Cys mutants were treated with carboxyatractyloside to lock the carrier in the cytosolic state or with bongkrekic acid to generate the matrix state, and then with the membrane-impermeable SH reagent eosin-5-maleimide (EMA) to probe accessibility. The amino acid residues S213C, L214C, F231C and L232C were not labeled, indicating that these 4 residues must have been buried in the membrane, whereas the region between residues K215 and S230 is accessible to labeling and must, therefore, have protruded into the aqueous phase. Residue L218C showed strong resistance against EMA labeling regardless of the state of the carrier, but the reason for such behavior is unclear. On the contrary, the labeling of the residues between F227C and S230C was strongly dependent on the state of the carrier. Thus, the C-terminal region of the second cytosolic loop in AAC2 changes its environment when the carrier cycles between the matrix and cytosolic state.     

Abundance,growth and grazing loss rates of picophytoplankton in Barguzin Bay,Lake Baikal

The abundance, growth, and grazing loss rates of picophytoplankton were investigated in August 2002 in Barguzin Bay, Lake Baikal. Water samples for incubation were taken once at a near-shore station and twice at an offshore station. Contributions of picophytoplankton to total phytoplankton were high (56.9–83.9%) at the offshore station and low (5.8–6.8%) at the near-shore station. The picophytoplankton community in the offshore station comprised mainly phycoerythrin (PE)-rich cyanobacteria, with eukaryotic picophytoplankton being less abundant. In contrast, as well as PE-rich cyanobacteria and eukaryotic picophytoplankton, phycocyanin (PC)-rich cyanobacteria were found in the near-shore station. At the offshore station, growth and grazing loss rates on 25 August were 0.56 and 0.43 day⁻¹, respectively, and on 29 August, 0.69 and 0.83 day⁻¹, respectively. At the near-shore station, growth and grazing loss rates were 1.61 and 0.70 day⁻¹, respectively. These results show that there is a difference in the abundance, composition, and ecological role in the microbial food web of picophytoplankton between the near-shore and the offshore areas in Barguzin Bay.     

Identification of (22R)-3 alpha,7 alpha,12 alpha,22- and (23R)-3 alpha,7 alpha,12 alpha,23-tetrahydroxy-5 beta-cholestanoic acids in urine from a patient with Zellweger's syndrome

The nature of two novel C27 bile acids present as the taurine conjugates in urine from a patient with Zellweger's syndrome was studied. Bile acids conjugated with taurine were isolated from unconjugated and glycine-conjugated bile acids by means of ion-exchange chromatography. After alkaline hydrolysis of the taurine conjugates, the hydrolysate was acidified and extracted with ether; the extract was again subjected to ion-exchange chromatography to separate neutral from acidic compounds. The neutral fraction, which consisted mainly of two steroidal lactones, was treated with lithium aluminum hydride, and the reduction products were identified as (22R)-5 beta-cholestane-3 alpha,7 alpha,12 alpha,22,26-pentol and (23R)-5 beta-cholestane-3 alpha,7 alpha,12 alpha,23,26-pentol by direct comparison of their gas-liquid chromatographic behaviors and mass spectral data with those of chemically synthesized authentic samples. Thus, the chemical structure of two native bile acids present in urine from a patient with Zellweger's syndrome should be formulated as (22R)-3 alpha,7 alpha,12 alpha,22-tetrahydroxy-5 beta-cholestanoic acid and (23R)-3 alpha,7 alpha,12 alpha,12 alpha,23-tetrahydroxy-5 beta-cholestanoic acid, respectively.     

Evaluation of interconversion between (R)- and (S)-enantiomers of β-aminoisobutyrate

The conversion of (R)- to (S)-β-aminoisobutyrate was observed in the presence of d-3-aminoisobutyrate-pyruvate aminotransferase, aminobutyrate aminotransferase, pyruvate and l-glutamate. The reverse reaction was also found in the presence of 2-oxoglutarate and l-alanine. Neither d-3-aminoisobutyrate-pyruvate aminotransferase nor aminobutyrate aminotransferase revealed a racemase activity of the enantiomorphs.     

Dense distribution of nuclear alkaline protease to nucleoli with increase in rapidly growing cells in rats

Neutral and alkaline proteases (chymotrypsin-like serine proteases) are tightly bound to chromatin in nuclei of various tissues of rats, and rapidly growing cells are abundant in the latter enzyme. Activities per mg DNA of these enzymes were measured with fractions of euchromatin, heterochromatin, nucleoli and extranucleoli from normal and regenerating livers, and Rhodamine sarcoma. With normal liver, both enzymes, respectively, were almost evenly distributed among the fractions, except that alkaline protease was significantly higher in nucleoli. The nucleolar alkaline protease increased by 30-60% with regenerating liver and by higher than 100% with the tumor.     

Stereospecific formation of (24E)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-en-26-oic acid and (24R,25S)-3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestan-26-oic acid from either (25R)- or (25S)-3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid by rat liver homogenate

Studies of the stereochemistry of the intermediates, 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholest-24-en-26-oic acid and 3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestan-26-oic acid, in the biosynthetic sequence between 3 alpha,7 alpha,12 alpha-trihydroxy-5 beta-cholestan-26-oic acid and cholic acid have been undertaken. (25R)- or (25S)-3 alpha,7 alpha, 12 alpha-Trihydroxy-5 beta-cholestan-26-oic acid was incubated with rat liver homogenates. The reaction products were converted to p-bromophenacyl ester derivatives and the esters were analyzed by high-performance liquid chromatography. By comparison with authentic samples of two (24E)- and (24Z)-isomers of the alpha, beta-unsaturated acid and of four isomers at C-24 and C-25 of the beta-hydroxy acid, (24E)-3 alpha,7 alpha, 12 alpha-trihydroxy-5 beta-cholestan-26-oic acid and (24R,25S)-3 alpha,7 alpha,12 alpha,24-tetrahydroxy-5 beta-cholestan-26-oic acid were found to be formed from either (25R)- or (25S)-3 alpha,7 alpha, 12 alpha-trihydroxy-5 beta-cholestan-26-oic acid. No formation of the (24Z)-isomer of the trihydroxycholestenoic acid or the other three isomers of the tetrahydroxycholestanoic acid was detected. The findings are discussed in relation to the assumed pathway for side chain cleavage in cholic acid biosynthesis.