Production of hepatitis delta virus and suppression of helper hepatitis B virus in a human hepatoma cell line.

The hepatitis delta virus (HDV) is a defective virus with a coat composing of the surface antigen of its helper virus, hepatitis B virus (HBV). Replication of HDV in the absence of HBV has been shown in cell cultures by transient transfection of the HDV plasmid. However, the formation and release of HDV virions have not been observed. In this report, a human hepatoma cell line HuH-7 was transiently cotransfected with HDV and HBV plasmids. The production of monomeric and multimeric antigenomic RNAs of HDV in the transfected cells indicated replication of the HDV genome. The major 3.5- and 2.1-kb RNAs of HBV were also expressed. Virions of both HDV and HBV were released from the cotransfected cells, as shown by the detection of monomeric genomic HDV RNA and partially double-stranded HBV DNA in the culture medium. Thus, this is the first report that describes the assembly and the release of HDV viral particles in an in vitro cell culture. The HDV virions released possessed physicochemical properties identical to those of the HDV virions found in infected human serum. Furthermore, expression of both the 3.5- and 2.1-kb RNAs of HBV was shown to be dramatically decreased by the presence of HDV, indicating suppression of the expression of HBV genes by HDV. The amount of HBV virions released was similarly suppressed by HDV. Cotransfection of HBV with an expression plasmid of the HDV delta antigen remarkably reduced the levels of the 3.5- and 2.1-kb HBV RNAs, indicating that suppression of the expression of HBV RNAs by HDV occurs via the action of the delta antigen. This HBV- and HDV-cotransfected human hepatoma cell line should provide an excellent system for the study of the function of the delta antigen and the interaction between HDV and its helper, HBV.     

Germin-Like Polypeptides Increase in Barley Roots during Salt Stress

The 26 kilodalton, isoelectric point 6.3 and 6.5 (Gs1 and Gs2) polypeptides that increase in barley (Hordeum vulgare L.) roots during salt stress were isolated and identified. Both Gs1 and Gs2 had high sequence similarity to germin, a protein that increases significantly in germinating wheat seeds. Like germin, Gs1 and Gs2 were resistant to proteases and were glycosylated. Immunoblots were probed with antibodies to Gs1 and Gs2 to determine the distribution of these polypeptides among organs and cell-free fractions. Gs1 and Gs2 were present in roots and coleoptiles, but absent from leaves. In roots, Gs1 and Gs2 were present in the mature region, but not the tip. Gs1 and Gs2 increased in roots, but decreased in coleoptiles in response to salt stress. Gs1 and Gs2 were distributed among the soluble, microsomal, and cell wall fractions of roots, but the majority of Gs1 and Gs2 was present in the soluble fraction. Although Gs1 and Gs2 were heat stable, their synthesis was not affected by abscisic acid treatment. Gs2 accumulated during abscisic acid treatment, whereas Gs1 did not. However, a 25.5 kilodalton, isoelectric point 6.1 polypeptide that was immunologically related to Gs1 did accumulate with abscisic acid treatment.     

西双版纳傣族“龙山”的生态学意义

傣族“龙山”是西双版纳地区傣族的民族植物文化中的一例。其概念是“神居住的地方”,在这个地方的动植物都是神的家园里的生灵,是神的伴侣,是不能砍伐、狩猎和破坏的。人     

中国小竹鼠生态的初步研究

小竹鼠在我国仅分布于云南西部热带亚热带地区。主要生活于山坡稀树灌丛、阔叶林、橡胶园及居民点附近。在盈江县分布于海拔300-950米地带。取食、休息、繁殖主要在洞道内。洞系由洞口、取食道、趋避道、窝及“厕所”组成。食物主要有棕叶芦、芦竹及三叶橡胶等18种,尤喜食橡胶树主根,因而对橡胶树危害很大。13号标本中雌7雄6。在盈江每胎2-3只,以2只为多。成年小竹鼠过独居生活,雌雄各有自己的洞系。     

Cross-linking of rabbit skeletal muscle troponin subunits: labeling of cysteine-98 of troponin C with 4-maleimidobenzophenone and analysis of products formed in the binary complex with troponin T and the ternary complex with troponins I and T

The sulfhydryl-specific, heterobifunctional, photoactivatable cross-linker 4-maleimidobenzophenone (BPMal) was used to study the interaction of rabbit skeletal muscle troponin subunits TnC, TnT, and TnI. TnC was labeled at Cys-98 by the maleimide moiety of BPMal and then mixed with either TnT alone or TnI plus TnT, in the presence of Ca2+. Upon photolysis, TnI and/or TnT formed covalent cross-links with TnC. The cross-linked TnC-TnT heterodimer obtained from the binary complex was digested into progressively smaller cross-linked peptides that were purified by HPLC and then characterized by amino acid analysis and sequencing. An initial cross-linked CNBr fraction contained the expected peptide CB9 (residues 84-135) of TnC, plus CNBr peptides spanning residues 152-230 of TnT. Results from a peptic digest of the CNBr cross-linked fraction permitted the identification of residues 159-197 as the most highly cross-linked region in TnT. A final subtilisin digest yielded a heterogeneous cross-linked fraction, which suggested that an especially high degree of cross-links was formed in the vicinity of residues 175-178 (Met-Lys-Lys-Lys) of TnT. Although this region of TnT had previously been implicated in binding, we show here for the first time that it is close to Cys-98 of TnC. In an analogous study on the binary complex of TnC and TnI [Leszyk, J., Collins, J. H., Leavis, P. C., & Tao, T. (1987) Biochemistry 26, 7042-7047], we previously showed that Cys-98 of TnC was cross-linked mainly to CN4, the "inhibitory region", of TnI.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serial propagation of mammalian cells on gelatin-coated microcarriers

The ability to serially propagate mammalian cells in microcarrier cultures is essential for large-scale operation. The success of such serial propagation depends on viable dissociation of cells from microcarriers and the normal growth and product formation after subsequent reinoculation. The high pH treatment developed for dissociating cells from DEAE-derivatized microcarriers was not as effective for a number of cell strains cultivated on gelatin-coated microcarriers. By prewashing the cell-laden microcarriers with buffer containing a chelating agent, bovine kidney cells, BK, human embryonic foreskin fibroblasts, FS-4, and continuous human kidney cells, TCL-598 which produces prourokinase, were viably dissociated from commercially available gelatin-coated microcarriers, Cytodex-3. Cells dissociated from microcarriers reattached and grew on micro-carriers subsequent to inoculation into subcultures. However, after subculturing, cells may attach at different rates to newly added beads and to conditioned microcarriers which cells had previously grown. It resulted in an uneven cell distribution on microcarriers and inferior growth kinetics. This effect was more profound for BK and FS-4 cells which are propagated with a low multiplication ratio. Specifically, BK cells attach to conditioned beads at a faster rate than to new beads, while FS-4 cells attach to new beads faster than to conditioned beads. Thus, for these two cell strains, a separator was used to separate the microcarriers from the suspension of dissociated cells before subsequent inoculation. For TCL-598 cells, which are propagated at a high multiplication ratio, this dissociation technique can be applied directly without the separation of dissociated cells and conditioned microcarriers. All the three cell lines tested exhibit normal growth kinetics in serial propagation on microcarriers. Furthermore, the production of prourokinase by TCL598 cells serially propagated on microcarriers was comparable to that inoculated from roller bottles.     

Phosphorylation of ankyrin decreases its affinity for spectrin tetramer

The effects of phosphorylation on the interaction between spectrin and ankyrin were investigated. Spectrin and ankyrin were phosphorylated using purified human erythrocyte membrane and cytosolic (casein kinase A) kinases. These two kinases have similar properties as well as activities toward spectrin and ankyrin. Both kinases catalyzed the incorporation of about 2 mol of phosphate/mol of spectrin and about 7 mol of phosphate/mol of ankyrin. These phosphates were incorporated primarily into seryl and threonyl residues of the proteins. The phosphopeptide maps of ankyrin phosphorylated by the membrane kinase and casein kinase A were identical. Binding studies indicate that ankyrin exhibits different affinities for spectrin dimers (KD = 2.5 +/- 0.9 X 10(-6) M) and tetramers (KD = 2.7 +/- 0.8 X 10(-7) M). These dissociation constants were not appreciably affected by the phosphorylation of spectrin. On the other hand, phosphorylation of ankyrin was found to significantly reduce its affinity for either phosphorylated or unphosphorylated spectrin tetramers (KD = 1.2 +/- 0.1 X 10(-6) M) but not spectrin dimers (KD = 2.5 +/- 0.4 X 10(-6) M). The same results were obtained using either the membrane kinase or casein kinase A as the phosphorylating enzyme. The above observation suggests that ankyrin phosphorylation may provide an important mechanism for the regulation of the erythrocyte membrane cytoskeletal network.     

Crassulacean Acid Metabolism and Crassulacean Acid Metabolism Modifications in Peperomia camptotricha

Peperomia camptotricha, a tropical epiphyte from Mexico, shows variable forms of Crassulacean acid metabolism (CAM). Young leaves exhibit CAM-cycling, while mature leaves show an intermediate type of metabolism, between CAM and CAM-cycling, having approximately the same amount of nighttime gas exchange as daytime. Metabolism of young leaves appears independent of daylength, but mature leaves have a tendency toward more CAM-like metabolism under short days (8 hours). Large differences in the physical appearance of plants were found between those grown under short daylengths and those grown under long daylengths (14 hours). Some anatomical differences were also detected in the leaves. Water stress caused a switch to CAM in young and mature leaves, and as water stress increased, they shifted to CAM-idling.