IgM rheumatoid factor autoantibody and immunoglobulin-producing precursor cells in the bone marrow of humans

The natures of the IgM rheumatoid factor (RF)-, IgM-, and IgG-secreting cells in the human bone marrow as compared to the peripheral blood, have been investigated by (1) response to the polyclonal B-cell activator, the Epstein-Barr virus (EBV), (2) sensitivity to the S-phase specific antimetabolite hydroxyurea, (3) presence of the BA-1 and Ia antigens on the cell surface, and (4) cell size, as determined by counter flow elutriation. The EBV-inducible bone marrow IgM-RF precursors derived from medium to large B cells that were inhibited by hydroxyurea pretreatment. The marrow total IgM response derived from small to medium size cells, and was only partially inhibited by hydroxyurea. Hydroxyurea had no effect on IgM-RF or IgM synthesis by peripheral blood cells. These results indicate that the marrow EBV-induced IgM-RF response is not representative of the response by peripheral blood cells, moreover; the marrow RF secreting response arises from a dividing cell pool that may represent newly generated autoreactive B cells.     

Genomic DNA sequence for human C-reactive protein

The gene for the prototype acute phase reactant, C-reactive protein, has been isolated from two lambda phage libraries containing inserted human DNA fragments using synthetic oligonucleotide probes. Nucleotide sequence analysis indicates that after coding for a signal peptide of 18 amino acids and the first two amino acids of the mature protein, there is an intron of 278 base pairs followed by the nucleotide sequence for the remaining 204 amino acids. The intron is unusual in that it contains on the positive strand a poly(A) stretch 16 nucleotides long and a poly(GT) region 30 nucleotides long which could adopt the Z-form of DNA. The nucleotide sequence reported here confirms the amino acid sequence of mature C-reactive protein as originally reported except that it codes for an additional 19 amino acids beginning at position 62. Thus DNA sequence analysis predicts that the mature protein consists of 206 amino acids rather than 187 as originally reported. The mRNA cap site is located 104 nucleotides from the start of the signal peptide and there is a 3' noncoding region 1.2 kilobase pairs in length. The gene has a typical promoter containing the sequences TATAAAT and CAAT 29 and 81 base pairs upstream, respectively, of the cap site.     

An improved DNA sequencing strategy

A modification of Hong's systematic DNA sequencing strategy is described. The original procedure has been simplified and transfectant yield increased. After DNase I limited cleavage in the presence of Mn2+, the single-cut linear DNA does not have to be separated from supercoiled or open circular DNA on an agarose gel. After ligation, the DNA is digested with a second restriction endonuclease for which a unique cleavage site resides between the insert and the first restriction endonuclease cutting site. The original intact DNA is linearized whereas the deleted subclone is not. The background is decreased to an undetectable level. This DNA sequencing strategy was tested on a 1.4-kb DNA fragment containing the araC regulatory gene from Erwinia carotovora. A set of subclones sufficient to sequence the fragment on both strands was produced in 2 days and the yield was at least 60-fold higher than in the original protocol.     

The placenta as a site of cytomegalovirus infection in guinea pigs.

The development of cytomegalovirus (CMV) infection in the placenta was studied in Hartley guinea pigs inoculated at midgestation, and its role in determining the outcome of fetal CMV infection was assessed. A hematogenous spread of CMV from the mother to the placenta occurred early during the course of the infection. However, the virus remained present in placental tissues long after CMV had been cleared from maternal blood (i.e., 3 and 4 weeks postinoculation). At that time, the virus was able to replicate in placental tissues in the presence of specific maternal antibodies. Viral nucleocapsids were seen within nuclei of trophoblastic cells, and virions were present surrounding infected cells. In addition, typical CMV-induced histopathological lesions bearing CMV antigens were consistently localized at the transitional zone between the capillarized labyrinth and the noncapillarized interlobium. Whenever CMV infection of the fetus occurred, virus was isolated from the associated placenta. Among placental-fetal units with CMV-infected placentas, only 27% of the fetuses were found to be infected. In addition, there was a delay in the establishment of the infection in the fetus in relation to the placenta, although frequencies of virus isolation in placental and fetal tissues peaked at 3 weeks after CMV inoculation. These results suggest that during primary CMV infection of pregnant guinea pigs, the placenta not only serves as a reservoir for CMV but also acts to limit transmission of the virus to the fetus.     

Diterpenoids from Caesalpinia pulcherrima

Three new furanoditerpenoids of the caesalpin-type have been isolated from the roots of Caesalpinia pulcherrima. The structures of these compounds, vouacapen-5α-ol, 6β-cinnamoyl-7β-hydroxy-vouacapen-5α-ol and 8,9,11,14-didehydrovouacapen-5α-ol, were elucidated through interpretation of their spectral data. Sitosterol was also obtained.     

Identification and characterization of an ATP.Mg-dependent protein phosphatase from pig brain

Substantial amounts of ATP.Mg-dependent phosphorylase phosphatase (Fc. M) and its activator (kinase FA) were identified and extensively purified from pig brain, in spite of the fact that glycogen metabolism in the brain is of little importance. The brain Fc.M was completely inactive and could only be activated by ATP.Mg and FA, isolated either from rabbit muscle or pig brain. Kinetical analysis of the dephosphorylation of endogenous brain protein indicates that Fc.M could dephosphorylate 32P-labeled myelin basic protein (MBP) and [32P]phosphorylase alpha at a comparable rate and moreover, this associated MBP phosphatase activity was also strictly kinase FA/ATP.Mg-dependent, demonstrating that MBP is a potential substrate for Fc.M in the brain. By manipulating MBP and inhibitor-2 as specific potent phosphorylase phosphatase inhibitors, we further demonstrate that 1) Fc.M contains two distinct catalytic sites to dephosphorylate different substrates, and 2) brain MBP may be a physiological trigger involved in the regulation of protein phosphatase substrate specificity in mammalian nervous tissues.     

Regulation of polyamine biosynthesis by antizyme and some recent developments relating the induction of polyamine biosynthesis to cell growth

This review considers the role of antizyme, of amino acids and of protein synthesis in the regulation of polyamine biosynthesis.The ornithine decarboxylase of eukaryotic ceils and ofEscherichia coli coli can be non-competitively inhibited by proteins, termed antizymes, which are induced by di-and poly- amines. Some antizymes have been purified to homogeneity and have been shown to be structurally unique to the cell of origin. Yet, the E. c o l i antizyme and the rat liver antizyme cross react and inhibit each other's biosynthetic decarboxylases. These results indicate that aspects of the control of polyamine biosynthesis have been highly conserved throughout evolution.Evidence for the physiological role of the antizyme in mammalian cells rests upon its identification in normal uninduced cells, upon the inverse relationship that exists between antizyme and ornithine decarboxylase as well as upon the existence of the complex of ornithine decarboxylase and antizyme in vivo. Furthermore, the antizyme has been shown to be highly specific; its Keq for ornithine decarboxylase is 1.4 x 1011 M-1. In addition, mammalian ceils contain an anti-antizyme, a protein that specifically binds to the antizyme of an ornithine decarboxylase-antizyme complex and liberates free ornithine decarboxylase from the complex. In B. coli , in which polyamine biosynthesis is mediated both by ornithine decarboxylase and by arginine decarboxylase, three proteins (one acidic and two basic) have been purified, each of which inhibits both these enzymes. They do not inhibit the biodegradative ornithine and arginine decarboxylases nor lysine decarboxylase. The two basic inhibitors have been shown to correspond to the ribosomal proteins S₂₀/L₂₆ and L₃₄, respectively. The relationship of the acidic antizyme to other known B. coli proteins remains to be determined.     

Regeneration of the arterial wall in microporous,compliant, biodegradable vascular grafts after implantation into the rat abdominal aorta

Summary The ultrastructure of a new type of vascular graft, prepared from a mixture of polyurethane (95 weight %) and poly-L-lactic acid (5 weight %), was examined six weeks after implantation into the abdominal aorta of rats. These microporous, compliant, biodegradable, vascular grafts function as temporary scaffolds for the regeneration of the arterial wall.Smooth muscle cells, covering the grafts, regenerated a neo-media underneath an almost completely regenerated endothelial layer (neo-intima). These smooth muscle cells varied in morphology from normal smooth muscle cells to myofibroblasts. They were surrounded by elastic laminae and collagen fibers.Macrophages, epithelioid cells, multinucleated giant cells, fibroblasts and capillaries were present in the disintegrating graft lattices. The epithelioid cells and multinucleated giant cells engulfed polymer particles of the disintegrating grafts.The regeneration of the endothelial and smooth muscle cells is similar to the natural response of arterial tissue upon injury. The presence of macrophages, epithelioid cells, multinucleated giant cells, fibroblasts and capillaries in the graft lattices resembles the natural response of tissue against foreign body implants. Both of these responses result in the formation of a neo-artery that possesses sufficient strength, compliance and thromboresistance to function as a small caliber arterial substitute.Supported by Grant nr. 82.042 from the Dutch Heart Foundation     

Cloning of the pectate lyase genes from Erwinia carotovora and their expression in Escherichia coli

A hybrid cosmid coding for pectate lyase (PL) activity was identified from an Erwinia carotovora genomic library by an immunological screening method. A 7-kb DNA fragment was identified which codes for three proteins identical in size to proteins with PL activity purified from E. carotovora culture supernatants. The three proteins had apparent Mrs of 41, 44 and 44 X 10(3) as estimated by SDS-PAGE. None of the PLs were exported from Escherichia coli strain HB101 but all were found in the periplasmic space. Plant tissue was macerated by the PLs made in E. coli.     

The araBAD operon of Salmonella typhimurium LT2. I. Nucleotide sequence of araB and primary structure of its product, ribulokinase

Hybrid plasmids containing the araBAD operon of Salmonella typhimurium LT2 were characterized by Southern blot and genetic analyses. The nucleotide sequence of araB was determined. The araB gene product, ribulokinase (EC 2.7.1.16), was purified and the results of amino acid composition analysis and partial amino acid sequence are in agreement with predictions from the DNA sequence. Ribulokinase is 569 amino acid residues long and has a calculated Mr of 61 793. Ribulokinase shares significant homology with xylulose kinase from Escherichia coli. Codon usage in the araB gene does not favor those codons which have intermediate codon-anticodon binding energy.