Induction of SerpinB2 and Th1/Th2 Modulation by SerpinB2 during Lentiviral Infections In Vivo

SerpinB2, also known as plasminogen activator inhibitor type 2, is a major product of activated monocytes/macrophages and is often strongly induced during infection and inflammation; however, its physiological function remains somewhat elusive. Herein we show that SerpinB2 is induced in peripheral blood mononuclear cells following infection of pigtail macaques with CCR5-utilizing (macrophage-tropic) SIV_mac239, but not the rapidly pathogenic CXCR4-utilizing (T cell-tropic) SHIV_mn229. To investigate the role of SerpinB2 in lentiviral infections, SerpinB2^−/− mice were infected with EcoHIV, a chimeric HIV in which HIV gp120 has been replaced with gp80 from ecotropic murine leukemia virus. EcoHIV infected SerpinB2^−/− mice produced significantly lower anti-gag IgG1 antibody titres than infected SerpinB2^+/+ mice, and showed slightly delayed clearance of EcoHIV. Analyses of published microarray studies showed significantly higher levels of SerpinB2 mRNA in monocytes from HIV-1 infected patients when compared with uninfected controls, as well as a significant negative correlation between SerpinB2 and T-bet mRNA levels in peripheral blood mononuclear cells. These data illustrate that SerpinB2 can be induced by lentiviral infection in vivo and support the emerging notion that a physiological role of SerpinB2 is modulation of Th1/Th2 responses.     

Monoclonal antibodies to antigens abnormally expressed in breast cancer

We report the production, screening, and characterization of ten murine monoclonal antibodies directed at antigens that are expressed abnormally in human breast tumors. Immunoperoxidase staining of frozen and fixed tissues shows the antigens to be present at low levels on the luminal membrane of normal breast cells and at high levels in the cytoplasm and surface membrane of breast tumor cells. The ten antibodies appear to recognize six different epitopes on the basis of their quantitative differences in reactivity against four antigen preparations, as measured by ELISA. Immunoblots show that eight of the ten antibodies recognize a 300,000 MW molecule from breast tumor preparations; six of these antibodies also react with a second molecule from the same tumor preparations of 280,000 MW. Seven antibodies react with an antigen from milk fat globule membrane of 330,000 MW. It therefore appears that the two molecules from tumor tissue and the one molecule from normal tissue share common epitopes. Selected antibodies were tested for reactivity against 25 primary breast tumors and 14 pairs of primary and metastatic breast tumors. Three antibodies have broad reactivity and stain more than 80% of primary tumors; the three other antibodies identify subsets of those tumors. Results of staining pairs of primary and metastatic lesions show that metastases continue to express antigens of the primary lesion in a high percentage of cells.     

Purification to homogeneity and partial amino acid sequence of a fragment which includes the methyl acceptor site of the human DNA repair protein for O6-methylguanine.

DNA repair by O6-methylguanine-DNA methyltransferase (O6-MT) is accomplished by removal by the enzyme of the methyl group from premutagenic O6-methylguanine-DNA, thereby restoring native guanine in DNA. The methyl group is transferred to an acceptor site cysteine thiol group in the enzyme, which causes the irreversible inactivation of O6-MT. We detected a variety of different forms of the methylated, inactivated enzyme in crude extracts of human spleen of molecular weights higher and lower than the usually observed 21-24kDa for the human O6-MT. Several apparent fragments of the methylated form of the protein were purified to homogeneity following reaction of partially-purified extract enzyme with O6-[3H-CH3]methylguanine-DNA substrate. One of these fragments yielded amino acid sequence information spanning fifteen residues, which was identified as probably belonging to human methyltransferase by virtue of both its significant sequence homology to three procaryote forms of O6-MT encoded by the ada, ogt (both from E. coli) and dat (B. subtilis) genes, and sequence position of the radiolabelled methyl group which matched the position of the conserved procaryote methyl acceptor site cysteine residue. Statistical prediction of secondary structure indicated good homologies between the human fragment and corresponding regions of the constitutive form of O6-MT in procaryotes (ogt and dat gene products), but not with the inducible ada protein, indicating the possibility that we had obtained partial amino acid sequence for a non-inducible form of the human enzyme. The identity of the fragment sequence as belonging to human methyltransferase was more recently confirmed by comparison with cDNA-derived amino acid sequence from the cloned human O6-MT gene from HeLa cells (1). The two sequences compared well, with only three out of fifteen amino acids being different (and two of them by only one nucleotide in each codon).     

Effects of TCDD on the EGF receptor of XB mouse keratinizing epithelial cells

TCDD was found to cause a marked inhibition of 125I-epidermal growth factor (EGF) binding to its receptor on the cell surface of XB mouse keratinizing epithelial cells (XB cells) cultured in vitro. The EC50 concentration was estimated to be on the order of 3 x 10(-11) M 24 hours after TCDD administration. As early as 12 hours after the addition of 10(-9) M of TCDD, XB cells showed signs of a decline in 125I-EGF binding levels. The level of such EGF receptor downregulation reached a maximum at 24 hours, continued until day 2, but completely recovered by day 3. This was accompanied by a rise in protein kinase activities, particularly those of the protein tyrosine kinases during the initial period of 6-24 hours. To test the hypothesis that the EGF receptors of the cells, by showing TCDD-induced symptoms of downregulation, actually are being activated and triggering EGF-like signals, we examined the effects of both TCDD and exogenously added EGF on cell morphology, colony formation degree of keratinization, the pattern of activation of protein kinases and de novo protein synthesis, and EGF receptor phosphorylation. Based on the similarity of cell responses to these between TCDD- and EGF-treated cells, we concluded that TCDD, directly or indirectly, causes activation of the EGF receptor. In contrast, 12-O-tetradencanoylphorbol-13-acetate (TPA), which is known to downregulate EGF receptors by blocking their protein tyrosine kinase, produced dissimilar end results. The balance of evidence support the notion that the action of TCDD in this cell line is tightly coupled to the activation of the EGF receptor and that one of the key consequences of such a biochemical change is that it signals these cells to commit to terminal differentiation.     

Molecular evolution of enzyme structure: Construction of a hybrid hamster/Escherichia coli aspartate transcarbamoylase

Summary Aspartate transcarbamoylase (ATCase, EC 2.1.3.2) is the first unique enzyme common to de novo pyrimidine biosynthesis and is involved in a variety of structural patterns in different organisms. InEscherichia coli, ATCase is a functionally independent, oligomeric enzyme; in hamster, it is part of a trifunctional protein complex, designated CAD, that includes the preceding and subsequent enzymes of the biosynthetic pathway (carbamoyl phosphate synthetase and dihydroorotase). The complete complementary DNA (cDNA) nucleotide sequence of the ATCase-encoding portion of the hamster CAD gene is reported here. A comparison of the deduced amino acid sequences of the hamster andE. coli catalytic peptides revealed an overall 44% amino acid similarity, substantial conservation of predicted secondary structure, and complete conservation of all the amino acids implicated in the active site of theE. coli enzyme. These observations led to the construction of a functional hybrid ATCase formed by intragenic fusion based on the known tertiary structure of the bacterial enzyme. In this fusion, the amino terminal half (the “polar domain”) of the fusion protein was provided by a hamster ATCase cDNA subclone, and the carboxyl terminal portion (the “equatorial domain”) was derived from a clonedpyrBI operon ofE. coli K-12. The recombinant plasmid bearing the hybrid ATCase was shown to satisfy growth requirements of transformedE. coli pyrB ⁻ cells. The functionality of thisE. coli-hamster hybrid enzyme confirms conservation of essential structure-function relationships between evolutionarily distant and structurally divergent ATCases.     

Importance of secondary structure for endothelin binding and functional activity.

ET-1[16-Phe] and ET-1[12-Pro] were prepared in order to investigate the importance of secondary structure of ET-1 for receptor binding and function. ET-1[16-Phe] displayed the greatest binding and contractile potency of the ET-analogs tested in rabbit pulmonary artery, rat aorta, and rat left atria. ET-1[12-Pro] also exhibited low nanomolar binding in these tissues but showed less contractile activity than ET-1[16-Phe] or ET-1. The results indicate that the helical region between residues Lys9 and Cys15 of ET-1 is not critical for receptor binding and functional activity. However, replacement of His16 with Phe altered the charge characteristics of the C-terminal region of ET-1 producing the most potent ET-1 analog yet reported.     

The translational stop signal: Codon with a context, or extended factor recognition element?

Wide ranging studies of the readthrough of translational stop codons within the last 25 years have suggested that the stop codon might be only part of the molecular signature for recognition of the termination signal. Such studies do not distinguish between effects on suppression and effects on termination, and so we have used a number of different approaches to deduce whether the stop signal is a codon with a context or an extended factor recognition element. A data base of natural termination sites from a wide range of organisms (148 organisms, 40000 sequences) shows a very marked bias in the bases surrounding the stop codon in the genes for all organisms examined, with the most dramatic bias in the base following the codon (+4). The nature of this base determines the efficiency of the stop signal in vivo, and in Escherichia coli this is reinforced by overexpressing the stimulatory factor, release factor-3. Strong signals, defined by their high relative rates of selecting the decoding release factors, are enhanced whereas weak signals respond relatively poorly. Site-directed cross-linking from the +1, and bases up to +6 but not beyond make close contact with the bacterial release factor-2. The translational stop signal is deduced to be an extended factor recognition sequence with a core element, rather than simply a factor recognition triplet codon influenced by context.