Picomole analysis of glutathione, glutathione disulfide, glutathione S-sulfonate, and cysteine S-sulfonate by high-performance liquid chromatography

A method for simultaneous detection of picomole quantities of glutathione (GSH), glutathione disulfide (GSSG), glutathione S-sulfonate (GSSO3H), and cysteine S-sulfonate (CYSSO3H) by high-performance liquid chromatography has been developed. Compounds are separated by anion-exchange chromatography using a citric acid buffer system, and then derivatized postcolumn using o-phthalaldehyde with 2-mercaptoethanol, heated to 70 degrees C, and detected by fluorescence. The compounds elute with retention times of 12.5 min for GSH, 27.5 min for CYSSO3H, 29.8 min for GSSG, and 33.0 minutes for GSSO3H, with detection limits of 10, 200, 10, and 50 pmol, respectively. Recoveries are 103% for GSH, 102% for GSSG, 100% for CYSSO3H, and 96% for GSSO3H. Determination of target compounds in cells is described.     

Amino acid sequence of Manduca sexta adipokinetic hormone elucidated by combined fast atom bombardment (FAB)/tandem mass spectrometry

Combination of Fast Atom Bombardment Tandem Mass Spectrometry with Amino Acid Analysis assigns the amino acid sequence of the Manduca sexta adipokinetic hormone as pGlu-Leu-Thr-Phe-Thr-Ser-Ser-Trp-GlyNH2. Similarities and differences with other invertebrate hormones and with mammalian glucagon are discussed.     

Fatigue of immature baboon cortical bone

Strain-controlled uniaxial fatigue and monotonic tensile tests were conducted on turned femoral cortical bone specimens obtained from baboons at various ages of maturity. Fatigue loading produced a progressive loss in stiffness and an increase in hysteresis prior to failure, indicating that immature primate cortical bone responds to repeated loading in a fashion similar to that previously observed for adult human cortical bone. Bone fatigue resistance under this strain controlled testing decreased during maturation. Maturation was also associated with an increase in bone dry density, ash fraction and elastic modulus. The higher elastic modulus of more mature bone meant that these specimens were subjected to higher stress levels during testing than more immature bone specimens. Anatomical regions along the femoral shaft exhibited differences in strength and fatigue resistance.     

Interleukins 2 and 3 regulate the in vitro proliferation of two distinguishable populations of 20-alpha-hydroxysteroid dehydrogenase-positive cells

The ability of interleukin 2 (IL 2), interleukin 3 (IL 3), and granulocyte/macrophage colony-stimulating factor (GM-CSF) to induce the proliferation of cells from thymus, spleen, or bone marrow was examined and compared with their ability to induce expression of the enzyme 20-alpha-hydroxysteroid dehydrogenase (20 alpha SDH). In the thymus, the peanut agglutinin agglutinated cells (PNA+) lacked 20 alpha SDH and showed no detectable response to IL 2, IL 3, or GM-CSF in either proliferation or induction of 20 alpha SDH. In contrast, the PNA nonagglutinated (PNA-) subpopulation expressed 20 alpha SDH and proliferated in response to Con A and/or IL 2. The responding cells that could be expanded in vitro with IL 2 expressed high levels of 20 alpha SDH. Neither IL 3 nor GM-CSF in the presence or absence of Con A had a demonstrable effect on the PNA- population. In cultures of bone marrow cells, both IL 3 and GM-CSF induced proliferation, whereas IL 2 had no effect on proliferation in the presence or absence of Con A. Thy-1-depleted bone marrow cells, expanded in tissue culture with IL3, contained cells that co-expressed Thy-1 and 20 alpha SDH. In contrast, cells proliferating in vitro to GM-CSF did not expressed Thy-1 or 20 alpha SDH. In cultures of normal splenic lymphocytes, two populations of cells capable of expressing 20 alpha SDH were detected. One population could be expanded in vitro with IL 2 and Con A, whereas the second was responsive to IL 3. In spleens from athymic mice, only the latter cells were detected. These results demonstrate that IL 3 and IL 2 responsiveness distinguishes two populations of 20 alpha SDH cells. The relevance of these observations to the possible relationship of IL 3 and IL 2 in T cell differentiation is discussed.     

Biosynthesis of riboflavin in Bacillus subtilis: origin of the four-carbon moiety.

We studied the incorporation of [1-13C]ribose and [1,3-13C2]glycerol into the riboflavin precursor 6,7-dimethyl-8-ribityllumazine, using a riboflavin-deficient mutant of Bacillus subtilis. The formation of the pyrazine ring requires the addition of a four-carbon moiety to a pyrimidine precursor. The results show that C-6 alpha, C-6, C-7, and C-7 alpha of 6,7-dimethyl-8-ribityllumazine were biosynthetically equivalent to C-1, C-2, C-3, and C-5 of a pentose phosphate. C-4 of the pentose precursor was lost through an intramolecular skeletal rearrangement. Thus, the last steps in the biosynthesis of 6,7-dimethyl-8-ribityllumazine apparently involve the same mechanism in bacteria as in fungi.     

Stepwise assembly of a pre-mRNA splicing complex requires U-snRNPs and specific intron sequences

We have investigated the early events of pre-mRNA splicing in vitro by sucrose gradient sedimentation analysis. Time course experiments revealed the assembly, in two steps, of a large (50S) pre-mRNA splicing complex, preceded by formation of two other complexes that sediment at approximately 22S and 35S. Pre-mRNA and the intermediates and products of the in vitro splicing reaction cosediment with the 50S complex, while only pre-mRNA is associated with the 22S and 35S complexes. No splicing is observed in the absence of a 50S complex. Formation of the 50S complex requires ATP, whereas formation of the 22S and 35S complexes does not. U-snRNPs are necessary for assembly of the 35S and the 50S complexes but not for assembly of the 22S complex. Analysis with mutant substrate RNAs demonstrated that a polypyrimidine stretch near the 3' splice site and an intact 5' splice site are absolutely required for splicing complex formation.     

Analysis of an activatable promoter: sequences in the simian virus 40 late promoter required for T-antigen-mediated trans activation.

The late promoter of simian virus 40 (SV40) is activated in trans by the viral early gene product, T antigen. We inserted the wild-type late-promoter region, and deletion mutants of it, into chloramphenicol acetyltransferase transient expression vectors to identify promoter sequences which are active in the presence of T antigen. We defined two promoter activities. One activity was mediated by a promoter element within simian virus 40 nucleotides 200 to 270. The activity of this element was detectable only in the presence of an intact, functioning origin of replication and accounted for 25 to 35% of the wild-type late-promoter activity in the presence of T antigen. The other activity was mediated by an element located within a 33-base-pair sequence (simian virus nucleotides 168 to 200) which spans the junction of the 72-base-pair repeats. This element functioned in the absence of both the origin of replication and the T-antigen-binding sites and appeared to be responsible for trans-activated gene expression. When inserted into an essentially promoterless plasmid, the 33-base-pair element functioned in an orientation-dependent manner. Under wild-type conditions in the presence of T antigen, the activity of this element accounted for 65 to 75% of the late-promoter activity. The roles of the 33-base-pair element and T antigen in trans-activation are discussed.     

Are Chaoborus larvae more abundant in acidified than in non-acidified lakes in Central Canada?

Eriksson et al (1980) hypothesized that the abundance of certain macroinvertebrate predators, such as larvae of the phantom midge Chaoborus , should increase in acidified lakes because of the elimination of fish. To examine the influence of pH and presence of fish on Chaoborus abundance, we surveyed Chaoborus populations in 33 lakes in Ontario, Canada which ranged in pH from 4.5 to 7.4. Chaoborus larvae were not more abundant in the acidified lakes that were devoid of fish than in the remaining lakes. Therefore, we concluded that pH and presence of fish are not prime determinants of total Chaoborus abundance in Canadian Shield lakes. We hypothesized that significant increases in Chaoborus abundance should only be anticipated when fish populations are eliminated by acidification of relatively nutrient rich lakes.     

MSI2通过下调circRNA_001214促进急性髓系白血病细胞HL60生长

Musashi-2（MSI2）是一种RNA结合蛋白质,对维持造血干细胞功能具有重要作用。研究表明,MSI2高表达能促进急性髓系白血病（acute myelocytic leukemia, AML）进展,但其作用机制尚不明确。本研究稳定沉默HL60细胞MSI2后,第1、2、3、4 d对照组的相对细胞生长率分别为1.931 ± 0.027、3.070 ± 0.073、4.017 ± 0.092和4.215 ± 0.246;敲减组分别为1.927 ± 0.035、2.564 ± 0.090、2.825 ± 0.097和3.223 ± 0.182,两组相比具有统计学差异,P<0.001;细胞凋亡明显增加(7.967% ± 0.698% vs 3.400% ± 0.322%., P<0.01);G₀/G₁期细胞比例明显增高（67.430% ± 4.390% vs. 50.360% ± 2.160%, P<0.01）;NUMB蛋白明显上调,LEF1明显下降。环状RNA（circular RNA, circRNA）芯片筛选和荧光定量PCR验证显示,MSI2沉默组circRNA_001214表达水平是对照组3.48倍。这一结果也在NALM6细胞得到证实。进一步用生物信息学分析,显示circRNA_001214最可能与miR-1273a、miR-1273e和miR 5095结合,进而影响参与细胞凋亡相关基因（CYCS、AKT1、BAX、TNFRSF10A、TNFRSF10D）、Wnt信号基因（WNT4、WNT2B、WNT7B、 DKK2、SFRP1、CSNKE1和LEF1）以及参与细胞代谢相关基因（RPE, PGAM4, PGAM1, TAT, CBS、RPE、SUCLG2、PGAM4、PGAM1和 IDNK）。总而言之,MSI2可能通过干扰circRNA_001214生成,减少靶miRNA对凋亡、Wnt信号及细胞代谢相关基因表达的影响,促进细胞生长。