Tissue distribution of rat angiotensinogen mRNA and structural analysis of its heterogeneity

The tissue distribution and the structural heterogeneity of the rat angiotensinogen mRNA have been investigated with the aid of a previously cloned cDNA as well as a genomic DNA for rat angiotensinogen as analytical probes. The angiotensinogen mRNA is expressed not only in the liver but also in various tissues including the brain, kidney, adrenal gland, ovary, and lung. The relative levels of the mRNA in the above tissues have been estimated to be 3-4, 20-30 (for the next three tissues), and around 100 times less than that in the liver, respectively. The mRNAs in both hepatic and extrahepatic tissues are encoded by a single gene in the rat genome. At least four different size classes of the angiotensinogen mRNA that start with a single 5' terminus and differ only in the lengths of their 3'-untranslated regions have been identified, and these multiple mRNA species are most likely generated by using the polyadenylation signals AAUAAA and AUUAAA found 10-30 nucleotides upstream from the four polyadenylation sites. Because the structures of these multiple mRNA species do not vary among the tissues of the liver, brain, and kidney, angiotensinogen synthesized locally is structurally identical to that produced in the liver and may have some biological roles independent of the circulating angiotensinogen, mainly derived from the liver. In addition, the sequence of the 5'-flanking region of the angiotensinogen gene has been determined, and some features common to other steroid hormone-responsive genes have been discussed.     

Isolation of a human cDNA for alpha 2-thiol proteinase inhibitor and its identity with low molecular weight kininogen

A lambda gt11 cDNA library containing DNA inserts prepared from human liver mRNA has been screened with an antibody to human alpha 2-thiol proteinase inhibitor that was isolated from fresh plasma. Eighteen positive clones were isolated from one million phage, and each was plaque purified. The cDNA insert of one of these phage was sequenced and shown to code for alpha 2-thiol proteinase inhibitor as identified by a partial amino acid sequence of the light chain of alpha 2-thiol proteinase inhibitor. This cDNA insert contained 1529 base pairs coding for the complete alpha 2-thiol proteinase inhibitor. It included 45 base pairs of 5' noncoding sequence, 1281 base pairs that code for pre alpha 2-thiol proteinase inhibitor, a stop codon, 160 base pairs of 3' noncoding sequence, and 40 base pairs of poly(A) tail. The noncoding sequence on the 3' end contained a potential recognition site (AATAAA) for processing and polyadenylation of precursor messenger RNA. The amino acid sequence of alpha 2-thiol proteinase inhibitor deduced from the cDNA showed a striking similarity (overall homology at 74%) to that of bovine low molecular weight (LMW) kininogen, including two internally repeated sequences and a nonapeptide sequence of bradykinin. These data clearly indicated that alpha 2-thiol proteinase inhibitor and LMW kininogen are identical. This was further supported by immunological cross-reactivity between alpha 2-thiol proteinase inhibitor and LMW kininogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytologic appearance of carcinosarcoma (malignant ameloblastoma and fibrosarcoma) of the maxilla. A case report.

The cytologic appearance of a carcinosarcoma (malignant ameloblastoma and fibrosarcoma) of the maxilla in a 63-year-old man is described. On his first admission the diagnosis of malignant ameloblastoma was made on biopsy. After five surgical excisions, radiotherapy and chemotherapy, the diagnosis was changed to carcinosarcoma because the stromal cells of the tumor had become malignant. Aspiration biopsy cytology of the tumor, with a cystic lesion found at the left suborbital area, revealed malignant epithelial cells, indicating malignant ameloblastoma. Imprint smears of both surgically resected and autopsy material showed two types of malignant neoplastic cells, of epithelial and mesenchymal origin.     

Response of Japanese beech (Fagus japonica Maxim.) sprouts to canopy gaps

The response of Japanese beech (Fagus japonica Maxim.) sprouts to canopy gaps in natural beech forest in central Japan was studied using two contrasted gaps in which tree-ring chronologies of regenerating stems were analyzed. The gaps were created by uprooting of a single Quercus mongolica var. grosseserrata stem (diameter: 50 cm; gap size: 40 m²; 23 years old) and by concurrent uprootings of four F. japonica stools (gap size: 180 m²; 30 years old). Japanese beech sprouts emerged before and after the gap formation and dominated stem populations in both gaps. In gaps, growth of F. japonica sprouts was equal or lower than growth of stems of seed origin, but most sprouts (F. japonica, Acer mono var. marmoratum) appeared a few years before emergence of seedlings. The small gap created by single stem fall was dominated by some beech sprouts from stools adjacent to the gap. The multiple gap was not closed by beech sprouts from stools surrounding the gap, but some dominant beech stems were resprouts from the uprooted beech stools. The existence of a sprout bank under the canopy may play an important role in the closing process of gaps in natural Japanese beech forest.     

A novel cytokine-inducible gene CIS encodes an SH2-containing protein that binds to tyrosine-phosphorylated interleukin 3 and erythropoietin receptors.

Cytokines manifest their function through alteration of gene expression. However, target genes for signals from cytokine receptors are largely unknown. We therefore searched for immediate-early cytokine-responsive genes and isolated a novel gene, CIS (cytokine inducible SH2-containing protein) which is induced in hematopoietic cells by a subset of cytokines including interleukin 2 (IL2), IL3, granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (EPO), but not by stem cell factor, granulocyte colony-stimulating factor and IL6. The CIS message encodes a polypeptide of 257 amino acids that contains an SH2 domain of 96 amino acids in the middle. To clarify the function of CIS in cytokine signal transduction, we expressed CIS in IL3-dependent hematopoietic cell lines under the control of a steroid-inducible promoter. The CIS product stably associated with the tyrosine-phosphorylated beta chain of the IL3 receptor as well as the tyrosine-phosphorylated EPO receptor. Forced expression of CIS by steroid reduced the growth rate of these transformants, suggesting a negative role of CIS in signal transduction. CIS induction requires the membrane-proximal region of the cytoplasmic domain of the EPO receptor as well as that of the common beta chain of the IL3, IL5 and GM-CSF receptor, whereas CIS binds to the receptor that is tyrosine phosphorylated by cytokine stimulation. Thus CIS appears to be a unique regulatory molecule for cytokine signal transduction.     

Isolation and characterization of a plaque-forming lambda bacteriophage carrying a ColE1 plasmid

A plaque-forming lambdaimm434 bacteriophage carrying the entire genome of colicinogenic factor E1 has been isolated and characterized. This phage, lambdaimm434ColE1, can lysogenize as a stable plasmid within a recombination-deficient Escherichia coli cell that lacks the normal attachment site for lambda phage. Furthermore, it has been found that lambdaimm434ColE1 phage carrying amber mutations in the O and P genes of the lambda genome, i.e., lambdaimm434OamPamColE1, behaves as a plaque-forming phage, and this finding suggests that the ColE1 factor DNA permits replication of the DNA of the plaque-forming phage.     

1H and 15N NMR study of human lysozyme.

The 15N signal assignment of human lysozyme was carried out by using 1H-1H and 1H-15N two dimensional experiments. To solve the severe overlap problem of the NH signals, uniform labeling of the protein with 15N was introduced. The uniformly 15N labeled protein was prepared using a high-expression system of Saccharomyces cerevisiae. From the analyses of 1H and 15N NMR spectra, all of the backbone 15N signals of the molecule were assigned to each specific residue in the amino acid sequence. Recently published proton signal assignments [Redfield & Dobson (1990) Biochemistry, 29, 7201-7214] were confirmed by these complementary data. In addition, assignments were extended to side chain 15NH2 groups of asparagine and glutamine. Elements of secondary structure were deduced from the pattern of sequential and medium-range NOE connectivities. Two beta-sheets and four alpha-helices could be identified in the protein, which were in good agreement with those determined by X-ray crystallography. The interaction between human lysozyme and its inhibitor N-acetyl-chitotriose was investigated by 15N-1H HMQC spectra. Most of the 15N-NH cross-peaks in the spectra were separated well enough to be followed during the titration experiment. Residues whose NH proton signals decrease in intensity upon complex formation, are located mainly around subsites B, C, and D. Local conformational changes were observed around the fourth helix adjacent to the cleft of human lysozyme.     

Inhibition of cell adhesion by high molecular weight kininogen

An anti-cell adhesion globulin was purified from human plasma by heparin-affinity chromatography. The purified globulin inhibited spreading of osteosarcoma and melanoma cells on vitronectin, and of endothelial cells, platelets, and mononuclear blood cells on vitronectin or fibrinogen. It did not inhibit cell spreading on fibronectin. The protein had the strongest antiadhesive effect when preadsorbed onto the otherwise adhesive surfaces. Amino acid sequence analysis revealed that the globulin is cleaved (kinin-free) high molecular weight kininogen (HKa). Globulin fractions from normal plasma immunodepleted of high molecular weight kininogen (HK) or from an individual deficient of HK lacked adhesive activity. Uncleaved single-chain HK preadsorbed at neutral pH, HKa preadsorbed at pH greater than 8.0, and HKa degraded further to release its histidine-rich domain had little anti-adhesive activity. These results indicate that the cationic histidine-rich domain is critical for anti-adhesive activity and is somehow mobilized upon cleavage. Vitronectin was not displaced from the surface by HKa. Thus, cleavage of HK by kallikrein results in both release of bradykinin, a potent vasoactive and growth-promoting peptide, and formation of a potent anti-adhesive protein.     

Cloning and nucleotide sequence of a human Zn-alpha 2-glycoprotein cDNA and chromosomal assignment of its gene

A cDNA clone of Zn-alpha 2-glycoprotein (Zn alpha 2gp) was isolated from a human prostate library. The amino acid sequence of prostate Zn alpha 2gp deduced from the nucleotide sequence was identical to the one previously reported on the Zn alpha 2gp protein purified from human blood plasma, except at three positions: the 65th and 222nd amino acid residues were Gln (----Glu) and Glu (----Gln), and there was a two amino acid insertion (Ile-Phe) between the 75th (Glu) and 76th (Met) amino acids. Southern blot analysis of human genomic DNA, however, suggested a single gene encoding Zn alpha 2gp. Using a panel of rodent-human somatic cell hybrids, the Zn alpha 2 gp gene was assigned to human chromosome 7.