Molecular cloning of transcripts that accumulate during the late G1 phase in cultured mouse cells

To identify previously undetected genes that might be involved in later stages of the transition from a quiescent state (G0) to the DNA synthetic phase (S) of murine cells, we set out to isolate cDNA clones derived from mRNAs that appear late in G1 phase in serum-stimulated cells. A lambda-cDNA library was prepared using poly(A)+ RNA from chemically transformed Balb/c 3T3 cells (BP/A31) that had been brought to quiescence and subsequently stimulated for 12 h with serum. From the first screening of approximately 21,000 recombinant phage plaques, about 100 clones were isolated that hybridized to a single-stranded cDNA pool derived from stimulated-cell RNA but not to DNAs made from resting-cell RNA. Eventually, six different clones were identified. The mRNAs from five of these genes increased gradually during the G0 to S transition, in contrast to the "immediate-early" rise of c-myc mRNA or the later rise of thymidine kinase mRNA. These six clones were sequenced and compared to the GenBank database. Clones LG-80, LG-2, and LG-69 are highly homologous to beta-actin, lactate dehydrogenase, and alpha-tubulin. Clones LG-5, LG-61, and LG-74 had no significant homologies to known sequences. A subtractive cDNA library was used to isolate two additional clones, Sub-S1 and Sub-S2; these have homologies to enolase and ribosomal protein L32. Additional studies that examine the function and regulation of these newly identified "late response" genes in the pre-DNA synthesis pathway are in progress.     

Molecular studies on trypanothione reductase, a target for antiparasitic drugs

Trypanosoma and Leishmania are parasitic protozoa that cause a variety of diseases, which include African sleeping sickness and oriental sore. Attempts to determine pharmaceutically exploitable differences between host and parasite biochemistry have identified the unique trypanothione pathway as a possible target. This pathway includes the enzyme trypanothione reductase, the parasite analogue of glutathione reductase.     

中国吻额蛛属一新种（蜘蛛目：皿蛛科：微蛛亚科）

本文描述了作者1986年采自湖北省神农架林区的吻额蛛属Aprifrontalia一新种:膨大吻额蛛,新种Aprifrontalia afflata sp.nov.。目前,除本新种外,世界仅报道过1种:Aprifrontalia mascula,本新种雄蛛最显著的特征是:其额向前突呈吻状,触肢胫节前端甚膨大。外雌器形成一斜向下方的突起。模式标本保存于白求恩医科大学生物教研室。     

Epitope mapping of snake venom phospholipases A2 with pseudexin monoclonal antibodies

Fifteen different monoclonal antibodies, developed against a pseudexin A, B, and C mixture, were screened for linear epitope recognition. Peptides (9-mers) spanning pseudexin B were synthesized on alanine-derivatized polyethylene pins and subsequently probed with antibody. Four antibodies recognized linear epitopes of pseudexin A, pseudexin B, and also nonidentical sequences found in other phospholipases A₂ (PLA₂s) as determined by enzyme-linked immunosorbent assays. Three antibodies recognized a highly conserved site important in calcium binding and the interlocking of dimeric forms of PLA₂. Antibodies neutralizing lethal or enzymatic effects of PLA₂ did not recognize linear epitopes.     

Rat cystathionine beta-synthase. Gene organization and alternative splicing.

We elucidated the structure and alternative splicing patterns of the rat cystathionine beta-synthase gene. The gene is 20-25 kilobase pairs long, and its coding region is divided into 17 exons. These are alternatively spliced, forming four distinct mRNAs (types I through IV). The predicted open reading frames encode proteins of 61.5, 39, 60, and 52.5 kDa, respectively. Exons 13 and 16 are used alternatively and mutually exclusively. Exon 13 includes a stop codon and encodes the unique carboxyl-terminal sequence found in types II and IV. Exon 16 is present only in type I. Types I and III, which differ by 42 nucleotides (exon 16), are the predominant synthase mRNA forms in rat liver. Seventeen arginine peptides from pure liver synthase matched the deduced amino acid sequences of types I and III. These two polypeptides are detectable in liver extracts; each exhibits enzymatic activity when expressed in transfected Chinese hamster cells. Synthase shows substantial sequence similarity with pyridoxal 5'-phosphate dependent enzymes from lower organisms. Similarity of synthase to Escherichia coli O-acetylserine (thiol)-lyase (cysK) is 52%; E. coli tryptophan synthase beta chain (trpB), 36%; yeast serine deaminase, 33%. Lysine 116 in synthase aligns with the established pyridoxyllysine residue of these enzymes suggesting that it is the pyridoxal 5'-phosphate binding residue.     

The influence of plant architecture on the foraging efficiencies of a suite of ladybird beetles feeding on aphids

By manipulating plant variety and predator species, we investigated the interactions of plant and predator traits in determining predation effectiveness. The predators were all coccinellid adults (Hippodamia convergens, Hippodamia variegata, Coccinella apunctata, and Coccinella septempunctata) and the prey were cabbage aphids (Brevicoryne brassicae). Foraging behavior of the four predators was observed on four crucifers that differed widely in their structures and surface textures (Brassica oleracea caulorapa, Brassica campestris, Brassica juncea crispifolia, and Hirschfeldia incana). Predation rates were significantly influenced by plant variety, a result we attribute to direct effects of plant morphology on predator mobility, falling frequency, and prey accessibility. Predation rates did not vary significantly among the ladybirds, although the four species did exhibit distinct foraging strategies as measured by time spent actively foraging, the rate of encountering aphids, and the fraction of aphids encountered that were consumed. The coccinellids also differed in their propensity for flying away from the plant, and in the frequency with which they fell from the plant. We did not detect any significant interaction effects between plant and predator species, suggesting that the main effects of plant and predator species may overwhelm their interactions in this kind of system. Our results suggest that the level of predation upon herbivorous insects may depend more upon plant architecture than on the particular species of natural enemies present.     

Mutational analysis of parasite trypanothione reductase: acquisition of glutathione reductase activity in a triple mutant

African trypanosomes contain a cyclic derivative of oxidized glutathione, N1,N8-bis(glutathionyl)spermidine, termed trypanothione. This is the substrate for the parasite enzyme trypanothione reductase, a key enzyme in disulfide/dithiol redox balance and a target enzyme for trypanocidal therapy. Trypanothione reductase from these and related trypanosomatid parasites is structurally homologous to host glutathione reductase but the two enzymes show mutually exclusive substrate specificities. To assess the basis of host vs parasite enzyme recognition for their disulfide substrates, the interaction of bound glutathione with active-site residues in human red cell glutathione reductase as defined by prior X-ray analysis was used as the starting point for mutagenesis of three residues in trypanothione reductase from Trypanosoma congolense, a cattle parasite. Mutation of three residues radically alters enzyme specificity and permits acquisition of glutathione reductase activity at levels 10(4) higher than in wild-type trypanothione reductase.     

Neoglycolipid analogues of ganglioside GM1 as functional receptors of cholera toxin.

We synthesized several lipid analogues of ganglioside GM1 by attaching its oligosaccharide moiety (GM1OS) to aminophospholipids, aliphatic amines, and cholesteryl hemisuccinate. We incubated GM1-deficient rat glioma C6 cells with each of the derivatives as well as native GM1 and assayed the cells for their ability to bind and respond to cholera toxin. On the basis of the observed increase in binding of 125I-labeled cholera toxin, it was apparent that the cells took up and initially incorporated most of the derivatives into the plasma membrane. In the case of the aliphatic amine derivatives, the ability to generate new toxin binding sites was dependent on chain length; whereas the C10 derivative was ineffective, C12 and higher analogues were effective. Increased binding was dependent on both the concentration of the neoglycolipid in the medium and the time of exposure. Cells pretreated with the various derivatives accumulated cyclic AMP in response to cholera toxin, but there were differences in their effectiveness. The cholesterol and long-chain aliphatic amine derivatives were more effective than native GM1, whereas the phospholipid derivatives were less effective. The distance between GM1OS and the phospholipid also appeared to influence its functional activity. The neoglycolipid formed by cross-linking the amine of GM1OS to phosphatidylethanolamine (PE) with disuccinimidyl suberate was less effective than the neoglycolipid formed by directly attaching GM1OS to PE by reductive amination. Furthermore, insertion of a C8 spacer in the former neoglycolipid rendered it even less effective.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estrogen-stimulation of postconfluent cell accumulation and foci formation of human MCF-7 breast cancer cells

Foci, nodules of cellular overgrowth, that appear after confluence are an in vitro characteristic of malignant transformation. A well-studied in vitro model of estrogen-dependent tumors is the MCF-7 cell line, derived from a pleural metastasis of a human breast adenocarcinoma. We report that cultivation of MCF-7 cells, using routine methods, results in extensive estrogen-stimulated postconfluent cell accumulation characterized by discrete three-dimensional arrays. Side view Nomarski optical sections revealed these to be principally multicellular foci with occasional domes and pseudoacinar vacuoles. This effect on MCF-7 cell growth occurs in media containing fetal bovine serum but not with calf serum or charcoal-dextran-treated fetal bovine serum unless supplemented with estrogens. Foci formation starts 5-6 days after confluence, and the number of foci generated is a function of the concentration of added estrogens. Foci formation is suppressed by the antiestrogens Tamoxifen and LY 156758. Addition of progesterone, testosterone, or dexamethasone had little or no effect, while various estrogens (ethinyl estradiol, diethylstilbestrol, and moxestrol) induced foci development. Clones derived from single cells of the initial MCF-7 population revealed a wide variance in estrogen-induced foci formation, demonstrating heterogeneity of this tumor cell line. The postconfluent cell growth of the estrogen receptor-deficient cell line, MDA-MB-231, contrasted with MCF-7 by developing an extensive multilayer morphology devoid of discrete structures. The tumorigenic potential of the MCF-7 cells used in our experiments was confirmed by their estrogen-dependent growth in immunosuppressed male BDF1 mice. These data suggest an estrogen receptor-based mechanism for the development of multicellular foci during postconfluent growth of MCF-7 cells. After confluence, foci, in contrast to the quiescent surrounding monolayer, retain proliferating cells. Focus formation, therefore, reflects the heterogeneous responsiveness of these cells to estrogens and should provide a model permitting in vitro comparisons between the progenitor cells of multicellular foci and the monolayer population.