Cloning of the gene encoding a catalytically self-sufficient cytochrome P-450 fatty acid monooxygenase induced by barbiturates in Bacillus megaterium and its functional expression and regulation in heterologous (Escherichia coli) and homologous (Bacillus megaterium) hosts

In a previous publication (Narhi, L. O., and Fulco, A. J. (1986) J. Biol. Chem. 261, 7160-7169) we described the characterization of a 119,000-dalton P-450 cytochrome that is strongly induced by barbiturates in Bacillus megaterium. In the presence of NADPH and O2, this single polypeptide can catalyze the hydroxylation of long-chain fatty acids without the aid of any other protein. The gene encoding this unique monooxygenase (cytochrome P-450BM-3) has now been cloned by an immunochemical screening technique. The Escherichia coli clone harboring the recombinant plasmid produces a 119,000-dalton protein that appears to be electrophoretically and immunochemically identical to the B. megaterium enzyme and contains the same N-terminal amino acid sequence. The recombinant DNA product also exhibits the characteristic cytochrome P-450 spectrum and is fully functional as a fatty acid monooxygenase. In E. coli, the synthesis of P-450BM-3 is directed by its own promoter included in the DNA insert and proceeds constitutively at a very high rate but is not stimulated by pentobarbital. However, when the cloned P-450BM-3 gene, either intact or in a truncated form, is introduced back into B. megaterium via an E. coli/Bacillus subtilis shuttle vector, its expression is constitutively repressed but is induced by pentobarbital. This finding demonstrates that the regulatory region of the P-450BM-3 gene that responds to barbiturates is included in the cloned DNA. The evidence also indicates that pentobarbital cannot directly act on the gene to cause induction but presumably interacts with another component such as a repressor molecule that is present in B. megaterium but is absent in the E. coli clone.     

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.     

Human thrombomodulin: complete cDNA sequence and chromosome localization of the gene

A human umbilical vein endothelial cell cDNA library in lambda gt11 was screened for expression of thrombomodulin antigens with affinity-purified rabbit polyclonal anti-thrombomodulin immunoglobulin G (IgG) and mouse monoclonal anti-human thrombomodulin IgG. Among 7 million recombinant clones screened, 12 were recognized by both antibodies. Two of these, lambda HTm10 and lambda HTm12, were shown to encode thrombomodulin by comparison of the amino acid sequence deduced from the nucleotide sequence to the amino acid sequence determined directly from tryptic peptides of thrombomodulin. Thrombomodulin mRNA was estimated to be 3.7 kilobases in length by Northern blot analysis of endothelial cell and placental poly(A)+ RNA. Thrombomodulin mRNA was not detected in human brain, HepG2 hepatoma cells, or the monocytic U937 cell line. Additional cDNA clones were selected by hybridization with the 1.2-kilobase insert of lambda HTm10. One isolate, lambda HTm15, contained a 3693 base pair cDNA insert with an apparent 5'-noncoding region of 146 base pairs, an open reading frame of 1725 base pairs, a stop codon, a 3'-noncoding region of 1779 base pairs, and a poly(A) tail of 40 base pairs. The cDNA sequence encodes a 60.3-kDa protein of 575 amino acids. The predicted protein sequence includes a signal peptide of approximately 21 amino acids, an amino-terminal ligand-binding domain of approximately 223 amino acids, an epidermal growth factor (EGF) homology region of 236 amino acids, a serine/threonine-rich segment of 34 amino acids, a membrane-spanning domain of 23 amino acids, and a cytoplasmic tail of 38 amino acids. The EGF-homology region consists of six tandemly repeated EGF-like domains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression and secretion of gro/MGSA by stimulated human endothelial cells.

Melanoma growth stimulatory activity factor (MGSA) is a polypeptide which was initially isolated from Hs294 human melanoma cells. Its sequence is identical to the deduced amino acid sequence of the human gro cDNA, isolated from a human tumor cell line. MGSA stimulates the proliferation of malignant melanoma cells, but its function for normal cells has not been defined. Here we report that human umbilical vein endothelial cells are capable of synthesizing and secreting MGSA. The expression and secretion of MGSA are strongly induced by factors often involved in inflammation such as IL-1, TNF, LPS and thrombin. The induction of MGSA mRNA is dose and time dependent and is independent of new protein synthesis. This stimulation could be mimicked by TPA, suggesting that the action could be mediated through activation of protein kinase C. Furthermore, addition of MGSA to the endothelial cell cultures induces gro/MGSA gene expression, implying that an autocrine mechanism exists. Our data suggest that the protein encoded by gro/MGSA mRNA may play a role in inflammation and exert its effects on endothelial cells in an autocrine fashion.     

Biosynthesis of fusarochromanone and its monoacetyl derivative by Fusarium equiseti.

One fluorescent compound previously named TDP-2 was isolated and purified from a rice culture of Fusarium equiseti (Alaska 2-2). Mass spectral and nuclear magnetic resonance data indicated that it is a C-3'-N-acetyl derivative of fusarochromanone, a newly discovered mycotoxin. Time course studies of synthesis of these two compounds on autoclaved rice and Czapek-Dox medium enriched with soybean peptone indicated that fusarochromanone was converted to TDP-2 in the cultures. A high concentration of peptone in the liquid medium may stimulate both fusarochromanone synthesis and its conversion to TDP-2.     

血革螨科二新种（蜱螨亚纲）

本文记述了血革螨科两新种:大理血革螨Haemogamasus daliensis sp.nov.和中华真厉螨Eulaelaps sinensis sp.nov.。该文对新种的形态特征作了描述,并与近似种进行了比较。     

喜马拉雅土拨鼠非冬眠期乳酸脱氢酶同工酶基因表达特征

用聚丙烯酰胺凝胶电泳和紫外光谱法分析非冬眠期喜马拉雅土拨鼠4种组织的乳酸脱氢酶(LDH)同工酶的酶谱及其活力,该鼠骨骼肌酶带的多态分布,可能是潜在的调节基因调控所致。另外,本文还对构象异构体产生的亚带进行了研讨。     

A reevaluation of the amino acid sequence of human follitropin beta-subunit

A collaborative study from two laboratories has been undertaken to re-evaluate the human follitropin -subunit sequence (hFSH), since areas of uncertainty remain in the wake of two earlier reports. The first report was by Shome and Parlow (1974). The second, by Saxena and Rathnam (1976), proposed revisions for sequence not definitively placed in the first study, as well as some differences in other placements. We have re-examined the sequence of the hFSH with more recent methodology. This has led to revision of certain areas of the sequence and resolution of differences between the two earlier proposals. Specifically, an-Ile-Ser- is established at 21–22, Asp at 41, Arg at 44, Lys at 46, and Glu at 111. These were areas of disagreement in the earlier proposals. A definitive placement of the residues around tryptophan-27 has now been obtained by three laboratories. C-terminal heterogeneity was observed with subunits ending at residue 107, 109, or 111. N-terminal heterogeneity has been observed in all preparations examined to date. A significant population of molecules with a proteolytic nick between residues 38–39 is noted. This is very likely an artifact of the collection and processing. The preparations examined in the present studies showed no evidence of residues 112–118 proposed by Saxena and Rathnam.     

Difference between amino acid residues in the metal-ligand environments of iron- and manganese-superoxide dismutases

Alignment of the amino acid sequences of the Pseudomonas ovalis and Photobacterium leiognathi iron-superoxide dismutases (Fe-SODs) with the known sequences of the manganese-superoxide dismutases (Mn-SODs) shows that both types of SOD are highly homologous (33-53% identity) and share residues for the metal coordination. The amino acid residues that form the environment of the metal ions appear to be also conserved between the Fe- and Mn-SODs, except that the Phe-84 and Gln-154 in the Mn-SODs are replaced by Tyr and Ala, respectively, in the Fe-enzymes. Since this latter residue contributes to formation of the hydrophobic metal-ligand environment through hydrogen bonding with Trp-133 and Tyr-34 in the Mn-SODs, its substitution by Ala should cause different micro environments between the metal centers of the Fe- and Mn-SODs. This difference may account for the metal specificity of both types of SODs demonstrated by previous reconstitution experiments.     

不同品种、不同花期的依兰花精油成分研究

为探讨依兰花精油的香气质量,我们对云南省西双版纳产依兰花精油以及引种栽培于西双版纳的泰国种、老挝种的依兰花精油的化学成分在Finnigan-4510 GC/MS/DS上用毛细管柱进行了定性和定量分析,鉴定了44个化学成分。分析结果表明,依兰油的香气与品种及精油中的酯类、醇类和酚醚的含量有关。倍半萜烯类和倍半萜醇类的含量越高,其香气质量越差。就西双版纳产依兰油的香气而言,依兰花由青转黄时得到的精油远较绿花和花蕾油为好。