Formation of lymphocyte colonies under serum-free culture conditions in normal individuals and patients with chronic lymphocytic leukemia

This paper describes a culture system which supports the formation of B cell and some T cell colonies under serum-free conditions in peripheral blood samples of normal individuals and patients with chronic lymphocytic leukemia (CLL) of B cell type. In this system, serum is replaced by bovine serum albumin, transferrin, cholesterol, insulin and catalase or horseradish peroxidase. In addition, it is necessary to add staphylococcus protein A, mitomycin-treated T cells as feeders and phytohemagglutinin leukocyte-conditioned medium as a source of growth factors. The plating efficiency is greatly enhanced when normal cells are incubated with galactose oxidase prior to plating and when CLL cells are exposed sequentially to neuraminidase and galactose oxidase.     

Identification of two segments, separated by approximately 45 kilodaltons, of the myosin subfragment 1 heavy chain that can be cross-linked to the SH-1 thiol

The thiol-specific photoactivatable reagent 4-(2-iodoacetamido)benzophenone (BPIA) can be selectively incorporated into the SH-1 of myosin subfragment 1 (S1), and upon photolysis an intramolecular cross-link is formed between SH-1 and the N-terminal 25-kDa region of S1. If a Mg2+-nucleotide is present during photolysis, cross-links can be formed either with the 25-kDa or with the central 50-kDa region [Lu, R. C., Moo, L., & Wong, A. G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6392-6396]. Heavy chains with these two types of intramolecular cross-links and un-cross-linked heavy chain have different mobility on sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels and therefore can be purified electrophoretically. Each type of heavy chain was cleaved with Staphylococcus aureus protease, chymotrypsin, or lysyl endopeptidase. The cleavage points were determined on the basis of the molecular weights of weights of peptides containing the N-terminus, which was identified with the use of an antibody. Locations of the cross-links were deduced by comparing the peptide maps of cross-linked and un-cross-linked heavy chains. The results indicate that the segment located about 12-16 kDa from the N-terminus of the heavy chain can be cross-linked to SH-1 via BPIA independently of the presence of a nucleotide, whereas the segment located 57-60 kDa from the N-terminus can be cross-linked to SH-1 only in the presence of a Mg2+-nucleotide. With use of the avidin-biotin system, it has been shown that SH-1 is located 13 nm from the head/rod junction [Sutoh, K., Yamamoto, K., & Wakabayashi, T. (1984) J. Mol. Biol. 178, 323-339]. Since BPIA spans less than 1 nm, our results show that two regions, separated by approximately 400 amino acid residues and located in the 25- and 50-kDa domains of S1, respectively, are also part of the head structure about 12-14 nm from the head/rod junction.     

水翁花蕾和水翁叶精油的化学成分研究

水翁的花营和鲜叶经水蒸汽蒸馏得到一种淡黄色的精油,前者出油率为0.18％。后者为0.08％。我们应用毛细管气相色谱,气相色谱/质谱联用,红外光谱和紫外光谱等方法,对两种精油进行化学分成分析,分别鉴定出35个和27个已知化学成分。两者相同的化学成分有:β-罗勒烯(Z)、β-罗勒烯(E)、α-蒎烯、β-蒎烯、月桂烯、小茴香烯、香叶醇、顺式-丁香烯、橙花叔醇等23个化学成分,分别占花营精油全油的90％和叶精油95％以上。     

Localization and Pattern of Graviresponse across the Pulvinus of Barley Hordeum vulgare

Pulvini of excised stem segments from barley (Hordeum vulgare cv `Larker') were pretreated with 1 millimolar coumarin before gravistimulation to reduce longitudinal cell expansion and exaggerate radial cell enlargement. The cellular localization and pattern of graviresponse across individual pulvini were then evaluated by cutting the organ in cross-section, photographing the cross-section, and then measuring pulvinus thickness and the radial width of cortical and epidermal cells in enlargements of the photomicrographs. With respect to orientation during gravistimulation, we designated the uppermost point of the cross-section 0° and the lowermost point 180°. A gravity-induced increase in pulvinus thickness was observable within 40° of the vertical in coumarin-treated pulvini. In upper halves of coumarin-treated gravistimulated pulvini, cells in the inner cortex and inner epidermis had increased radial widths, relative to untreated gravistimulated pulvini. In lower halves of coumarin-treated pulvini, cells in the central and outer cortex and in the outer epidermis showed the greatest increase in radial width. Cells comprising the vascular bundles also increased in radial width, with this pattern following that of the central cortex. These results indicate (a) that all cell types are capable of showing a graviresponse, (b) that the graviresponse occurs in both the top and the bottom of the responding organ, and (c) that the magnitude of the response increases approximately linearly from the uppermost point to the lowermost. These results are also consistent with models of gravitropism that link the pattern and magnitude of the graviresponse to graviperception via statolith sedimentation.     

Thrombolysis using liposomal-encapsulated streptokinase: an in vitro study

The clot-lysing ability of streptokinase (SK) was examined using membrane-bound thrombi. Encapsulation of SK in large unilamellar phospholipid vesicles (liposomes) resulted in entrapping approximately 30% of its original activity. Measurements of streptokinase activity for liposomal-encapsulated streptokinase (LESK) indicated little loss of activity or leakage in Tris-buffered saline over a 24-hr period at temperatures of 4 and 23 degrees C. However, incubation of free SK and LESK in platelet-poor plasma (PPP) at 37 degrees C resulted in a decrease of SK activity. The retention of SK activity in LESK was considerably higher than that of unentrapped SK. Clot-dissolving time (CDT) was measured by monitoring the pressure drop during slow filtration in plasma through membrane-bound thrombi. The results indicated that both LESK and free SK were able to activate the fibrinolytic system. Without prior incubation in PPP at 37 degrees C, the CDT of a SK and PPP mixture (SK/PPP) was 10.7 +/- 1.9 min (n = 12), while that of a LESK and PPP mixture (LESK/PPP) was 12.4 +/- 1.7 min (n = 12). The CDT-detected clot-lysing abilities of both SK and LESK were diminished by incubation in PPP, but to different extents. After 15- and 30-min incubations, the CDT of SK/PPP increased significantly to 15.5 +/- 1.5 and 24.1 +/- 2.4 min (n = 5, P less than 0.05), respectively. In contrast, the CDT of LESK/PPP increased to 13.3 +/- 0.8 min (n = 5) after 15 min of incubation and to 16.0 +/- 1.1 min (n = 5, P less than 0.05) after a 30-min incubation. These results suggest that entrapment of SK in liposomes preserves the thrombolytic potential of the plasminogen activator by limiting its exposure to the components of the plasma.     

Disulfide and secondary structures of recombinant human granulocyte colony stimulating factor

Molecular characteristics and secondary structures of recombinant methionyl human granulocyte colony stimulating factor produced by genetically engineered Escherichia coli are described. Limited radiolabeling of the protein with tritiated iodoacetate and determination of the labeled residue revealed that this recombinant protein contains only one free cysteine at position 17 which is not essential for activity. The free cysteine is inaccessible to modification unless the molecule is unfolded under denaturing conditions. The molecule forms two disulfide bridges which were assigned as Cys(36)-Cys(42) and Cys(64)-Cys(74) based on the results of isolation and characterization of disulfide-containing peptides obtained from a subtilisin digest of the intact protein. CD analyses and secondary structure prediction suggest that the molecule is abundant in alpha-helical structures.     

Disulfide structures of human interleukin-6 are similar to those of human granulocyte colony stimulating factor

The amino acid sequences of human interleukin-6 and granulocyte colony stimulating factor are approximately 30% homologous in the N-terminal region. The relative positions of four half-cystines in human interleukin-6 (IL-6) match four of the five in human granulocyte colony stimulating factor. Labeling experiments of recombinant interleukin-6 with tritiated iodoacetate confirmed that the molecule forms two intramolecular disulfide bonds and contains no detectable level of free sulfhydryls. By isolation and characterization of tryptic and subtilytic peptides obtained from different proteolytic digestions, the disulfide bonds of the IL-6 molecule were assigned to Cys44-Cys50 and Cys73-Cys83. The two disulfide bridges form two small loops which are separated by 22 amino acids. These structures are similar to those of recombinant granulocyte colony stimulating factor.     

Altered hepatic microsomal function and elevated protooncogene expression as residual effects in rats exposed to delta-9-tetrahydrocannabinol

The microsomal activation of the potent hepatocarcinogen aflatoxin B1 (AFB1) and the expression of selected protooncogenes were investigated in the livers of rats exposed to delta 9-tetrahydrocannabinol (THC). At equimolar levels of cytochrome P-450, the microsome-mediated binding of AFB1 to DNA was significantly lower (56% of the controls) in preparations from drug exposed rats. Hepatic expression of the c-k-ras protooncogene was 3-fold higher in THC exposed animals. These results suggest the possible occurrence of long lasting residual effects in the rats exposed to THC.     

Glutamic acid-88 is close to SH-1 in the tertiary structure of myosin subfragment 1

The thiol-specific photoactivatable reagent benzophenone iodoacetamide (BPIA) can be selectively incorporated into the most reactive thiol, SH-1, of myosin S1, and upon photolysis, an intramolecular cross-link is formed between SH-1 and the N-terminal 25-kDa region of S1. If a Mg2+-nucleotide is present during photolysis, cross-links can be formed either with the 25-kDa region or with the central 50-kDa region [Lu et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6392]. Comparison of the peptide maps of cross-linked and un-cross-linked S1 heavy chains indicates that the segment located about 12-16 kDa from the N-terminus of the heavy chain can be cross-linked to SH-1 via BPIA independently of the presence of a nucleotide whereas the segment located 57-60 kDa from the N-terminus can be cross-linked to SH-1 only in the presence of a Mg2+-nucleotide [Sutoh & Lu (1987) Biochemistry 26, 4511]. In this report, S1 was labeled with radioactive BPIA, photolyzed in the absence of nucleotide, and then degraded with proteolytic enzymes. Peptides containing cross-links were isolated by liquid chromatography and subjected to amino acid sequence analyses. The results show that Glu-88 is the major site and Asp-89 and Met-92 are the minor sites involved in cross-linking with SH-1 (Cys-707) via BPIA. These residues are very near the reactive lysine residue (Lys-83) but relatively remote in the primary structure from the putative nucleotide binding region.