Natural zeolite clinoptilolite increases the concentrations of sphingoid bases in the yeast Yarrowia lipolytica

In the present paper, we studied the effect of natural zeolite clinoptilolite on sphingolipid metabolism in the yeast Yarrowia lipolytica. We also investigated if zeolite addition had any impact on cell shape and size, as well as on the pH alterations during the culture growth. High performance liquid chromatography analysis of sphingoid bases obtained by acid hydrolysis of complex sphingolipids from Y. lipolytica showed that their concentrations markedly rose upon the zeolite addition. The largest increase among the identified molecular species of sphingoid bases was seen in C18 phytosphingosine, whose levels rose 6.2-fold and 22.3-fold after culturing cells for 24 and 36 hours respectively in the presence of finely ground zeolite. pH measurements of the culture medium showed a similarity between pH profiles of control and zeolite-supplemented cells, suggesting that ion-exchange capacity was not probably responsible for the observed change in sphingolipid metabolism. Scanning electron microscopy revealed that zeolite affected cell size and shape. Y. lipolytica cells grown in the absence of zeolite were oval-shaped with an average cell size of 0.7-2.7 microns, whereas when cultured with zeolite, they were round-shaped and larger, having an average cell size of 1.3-2.9 microns.     

Carbonic anhydrase gene expression in CA II-deficient (Car2−/−) and CA IX-deficient (Car9−/−) mice

Using real-time PCR and immunohistochemistry, we have examined the expression of carbonic anhydrase isozymes (CA) I, II, III, IV, IX, XII, XIII and XIV in the brain, kidney, stomach and colon of the wild-type, CA II-deficient ( Car2^−/− ), and CA IX deficient ( Car9^−/− ) mice. The expression of Car4, Car12, Car13 and Car14 mRNAs did not show any significant deviations between the three groups of mice, whereas both groups of CA deficient mice showed decreased expression levels of Car1 in the colon and Car3 in the kidney. The Car2 mRNA level was greatly reduced but not completely abolished in all four tissues from the Car2^−/− mice in which no CA II protein was expressed. Sequencing the Car2 cDNA isolated from C57BL6 Car2^−/− mice revealed two nucleotide differences from the wild-type C57BL6 mice. One is a silent polymorphism found in Car2 mRNA from wild-type DBA mice, which is the strain that provided the original mutagenized chromosome. The second change is a mutation that causes prematurely terminated translation at codon 155 (Gln155X). Car9 mRNA and CA IX protein expression levels were up-regulated about 2.5- and 3.6-fold, respectively, in the stomach of the Car2^−/− mice. These results suggest that the loss of function of cytosolic CA II in the stomach of Car2^−/− mice leads to up-regulation of an extracellular CA, namely CA IX, which is expressed on the cell surface of the gastric epithelium.     

Morphological and functional differences in the elbow extensor muscle between highly trained male and female athletes

The purpose of this study was to investigate the difference in the morphological and functional aspects of the triceps brachii muscle between highly trained male and female athletes who were members of the 1996 Japanese Olympic teams in each of three different events: soccer, gymnastics and judo. The thickness (TB_mt) and fibre pennation angle (TB_pen) of the triceps brachii muscle and force output during elbow extensions were determined using a B-mode ultrasound apparatus and an isokinetic dynamometer, respectively. The TB_mt and its value relative to upper arm length (TB_mt/l _ua) were significantly larger in the men than in the women in all the events except judo. In all the subjects, a significant correlation was found between TB_mt/l _ua) and TB_pen (r= 0.721, P < 0.05). The existence of the sex difference in TB_pen within the same event was in agreement with that observed in TB_mt/l _ua except for the soccer players. The TB_pen of the soccer players were similar in both sexes although a significant sex differences was found in TB_mt/l _ua. The isokinetic forces measured using the two velocities 60°· s^?1 (F ₆₀) and 180°· s^?1 (F ₁₈₀) were significantly correlated to the cross-sectional area (CSA) of the triceps brachii muscle estimated from TB_mt (r= 0.702, P < 0.05 for F ₆₀, and r= 0.776, P < 0.05 for F ₁₈₀). No significant sex differences were found in either F ₆₀/CSA or F ₁₈₀/CSA in any of the events. From these results, it could be assumed, at least in the Olympic athletes tested in this study, that the fibre angulation of the triceps brachii muscle was almost the same in the two sexes if allowance was made for the difference in the muscle size, and the sex difference in force generation capability of the triceps brachii muscle could in the main be attributed to the difference in CSA rather than in the architectural characteristics.     

Mechanisms responsible for delayed and immediate hemolytic transfusion reactions in a patient with anti-E + Jk(b)+ Di(b) and anti-HLA alloantibodies

Immediate hemolytic transfusion reactions (IHTR) occurred in the course of delayed hemolytic transfusion reactions (DHTR). An 84-year-old man had received a blood transfusion 20 years ago. Progressive anemia developed, because of continuous bleeding from a bladder tumor. He was transfused with concentrated red blood cells (CRC) which were Rh-E antigen negative, because he had anti-E antibodies (day 0). He received CRC on day 3, and underwent resection of bladder tumor on day 6. Although crossmatch-compatible CRCs were prepared for the operation, those were not required and were kept in a refrigerator in the ward. On day 9, when a CRC kept in the ward was transfused, he suddenly had a IHTR. In order to analyze a mechanism of IHTR, the anti-Jk(b) and anti-Di(b) antibodies, anti-HLA antibodies and the concentrations of inflammatory cytokines were measured in serum samples. The anti-Jk(b) and anti-Di(b) antibodies increased prior to IHTR experienced on day 9. The concentrations of IL-6 and IL-1beta increased from day 2, while the concentration of IL-8 increased from day 7. The anti-HLA class I antibody could be detected 2 days before IHTR. Thus, the anti-Jk(b) and anti-Di(b) antibodies induced the production of inflammatory cytokines and symptoms of DHTR and IHTR. The anti-HLA class I antibody could be produced in spite of using the filer for removing leukocytes, and may take part in the induction of IHTR. Further, blood products should be transfused soon after completing a crossmatch test in patients with anti-RBC alloantibodies.     

In vitro trypanocidal activity of dibutyltin dichloride and its fatty acid derivatives

Searching for new compounds against pathogenic trypanosomes has been substantially accelerated by the development of in vitro screening assays. In an attempt to explore the chemotherapeutic potential of organotin compounds and to broaden the search for newer trypanocides, fatty acid derivatives of dibutyltin dichloride were synthesized and their in vitro trypanocidal profiles studied on Trypanosoma brucei brucei, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. A 24-h time course experiment was conducted with various concentrations of the compounds using a 24-well microtiter plate technique. The compounds tested were trypanocidal in a dose-dependent fashion: inhibiting survival and growth, resulting in irreversible morphological deformation and the eventual death of the parasites. The minimum inhibitory concentrations of the tested diorganotins are at low micromolar ranges: from 0.15–0.75 M for T. b. brucei, T. b. gambiense and T. b. rhodesiense. These observations suggest that organotin has chemotherapeutic potential.     

Live varicella vaccine polarizes the mucosal adjuvant action of cholera toxin or its B subunit on specific Th1-type helper T cells with a single nasal coadministration in mice

This study was undertaken to determine whether the specific Th1- or Th2-cell response to varicella-zoster virus was induced predominantly by a mucosal adjuvant, cholera toxin, in mice. A commercially available live varicella vaccine (Oka strain) and cholera toxin or its B subunit were administered simultaneously via the nasal route. Delayed-type hypersensitivity to the Oka vaccine was induced, but the systemic neutralizing antibody response was low. The delayed-type hypersensitivity evoked after a single administration was relatively higher than that on administration three times. When spleen cells from mice immunized once with the vaccine and cholera toxin or its B subunit were restimulated with the live vaccine in vitro, there was greater thymidine uptake and production of interleukin- 2 (IL-2) than controls, but only a low level of IL-4 production. The production of IL-2 induced by the B subunit of cholera toxin was less than that by cholera toxin and a mutant of Escherichia coli enterotoxin on co-immunization with the vaccine in mice. Cholera toxin and its B subunit have been reported to induce predominantly a specific Th2-type T-cell response to various antigens. However, the Oka vaccine is an antigen that polarizes the activation of specific Th1/Th2-type T cells by cholera toxin or its B subunit to the Th1-type side. Cholera toxin and its B subunit are thus useful mucosal adjuvants for inducing cellular immunity to the Oka vaccine similar to Escherichia coli enterotoxin.     

Immunohistochemically demonstrated variation in expression of cathepsin E between uracil-induced papillomatosis and N-butyl-N-(4-hydroxybutyl)nitrosamine-induced preneoplastic and neoplastic changes in rat urinary bladder

Expression of rat urinary bladder cathepsin E in benign papillomatosis induced by uracil and various stages of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced carcinogenesis was investigated immunohistochemically. Seven-week-old, male F344/DuCrj rats were used. In the normal urothelium of control rats, cathepsin E stained in all layers of cells, although in umbrella cells and some basal cells the reaction was relatively weak. In rats given a diet containing 3% uracil for 5 weeks immunoreactivity of cathepsin E in uracil-induced papillomatosis was consistently homogeneous in all layers, but weaker than in normal urothelium. In rats given 0.05% BBN in drinking water for 12 weeks and subsequently maintained without treatment for 48 weeks cells with little cathepsin E, never observed in normal urothelium, appeared at 5 weeks above the basement membrane in the earliest stage of BBN-induced urinary bladder cancer (simple hyperplasia). Throughout the neoplastic process, groups of cells with a little cathepsin E were randomly distributed, with expression in the urothelium being markedly unstable. Almost all areas of squamous cell proliferation in TCC were negative for cathepsin E. Instability of cathepsin E expression in rat urothelium therefore appears characteristic for carcinogenesis and offers the possibility of using this feature as an early biomarker for urinary bladder carcinogenesis.     

Expression and localization of heat shock proteins in rat basophilic leukemia cells: differential modulation by degranulation,thermal or oxidative stress

BACKGROUND: Rat basophilic leukemia (RBL-2H3) cells are well characterized in terms of morphological and biochemical changes upon activation, and have been extensively used as a model system for studying the mechanisms of the immediate hypersensitivity reaction. To investigate whether overexpression of heat shock/stress proteins (HSP) is involved in the mast cell-dependent reactivity, we examined the adaptive responses of RBL-2H3 cells to classical stress conditions such as heat shock or oxidative injury produced by an aqueous extract of tobacco smoke. METHODS: HSP were determined by flow cytometry and immunocytochemistry. Degranulation was confirmed as the release of beta-hexosaminidase, determined spectrophotometrically, and by electron microscopy experiments. RESULTS: We found that RBL-2H3 cells respond to heat shock or oxidative injury by the synthesis of both the inducible 72 kDa HSP (Hsp70), and the oxidation-specific 32 kDa heme oxygenase (HO)-1. Heat shock induced mainly Hsp70 in a cell growth-dependent manner, whereas oxidative stress induced mainly HO-1 in a cell growth-independent manner. However, heat shock or oxidative stress had no significant effects on degranulation. CONCLUSION: Stress-mediated synthesis of HSP was not associated with RBL-2H3 degranulation and likewise, degranulation did not induce HSP.