Use of lacZ fusions to measure in vivo expression of the first three genes of the Escherichia coli unc operon.

We have constructed in-frame lacZ protein fusions to the first three genes of the Escherichia coli unc operon, which codes for the subunits of the proton-translocating ATPase. We have used these constructions to measure the relative in vivo expression of these genes. The second and third genes, uncB and uncE, which code for the a and c subunits of the F0 sector, were expressed at relative levels of approximately 1:10, although the measured expression of uncB depended upon how much of the gene was fused to lacZ. These rates compared favorably with the relative numbers of a and c subunits (a1:c10) in the purified F1F0 complex. The in vivo expression of uncI, the first gene of the operon, was very low, at best 10 to 20 times less than the expression of uncB.     

The primary structure of a protein carboxyl methyltransferase from bovine brain that selectively methylates L-isoaspartyl sites

Protein L-isoaspartyl methyltransferase (PIMT) transfers the methyl group of S-adenosyl-L-methionine to free alpha-carboxyl groups of atypical L-isoaspartyl residues in proteins. The complete primary structure of the type I isoform of bovine brain PIMT was determined by sequence analysis of peptides generated by endoprotease Lys-C, trypsin, cyanogen bromide, and endoprotease Asp-N digests. The correct composition of every peptide was verified by fast atom bombardment mass spectrometry. The efficiency of sequencing by tandem mass spectrometry was examined for several peptides by comparing its speed and accuracy with automated Edman degradation. Tandem mass spectrometry was used to determine the structure of the NH2-terminal blocked peptide derived from a hydroxylamine cleavage. PIMT is 226 residues with Mr = 24,500 and contains acetyl alanine as the amino-terminal residue. The partial sequence (141 residues from 8 tryptic peptides) of a homologous human red cell PIMT (Gilbert, J. M., Fowler, A., Bleibaum, J., and Clarke, S. (1988) Biochemistry 27, 5227-5233) shows a 97% identity with the corresponding peptides of the bovine brain enzyme. The complete brain enzyme sequence reported here bears no significant homology to any other known class of methyltransferase including those which methylate the side chain gamma-carboxyl group of receptor proteins involved in bacterial chemotaxis.     

The effects of xylazine and alpha-adrenoreceptor antagonists on bovine uterine contractility in vitro

The effects of alpha-adrenoreceptor antagonists prazosin (alpha-1), yohimbine (alpha-2), and idazoxan (alpha-2) on xylazine-induced bovine uterine contractility were tested in vitro. Uterine strips from proestrous/estrous and diestrous cows were mounted in tissue baths containing Tyrode's solution. Changes in uterine contractility were measured by strain gauge. The following results were observed: 1) Xylazine increased uterine contractility in a dose dependent manner (cumulative concentrations: 10(-8), 3x10(-8), 10(-7), 3x10(-7) and 10(-6)M). 2) Idazoxan (10(-8), 10(-7) and 10(-6)M) and yohimbine (10(-6), 10(-5) and 10(-4)M) antagonized uterine contractility induced by xylazine in a dose-dependent manner. Idazoxan was approximately 50 to 100 times more potent than yohimbine. 3) Prazosin (10(-5)M) did not alter the effect of xylazine on uterine contractility. These results suggested that xylazine-induced uterine contractility in the cyclic cow is directly mediated by myometrial alpha-2 adrenoreceptors.     

Cytogenetic characterization of 20 lymphoblastoid lines derived from human individuals differing in bleomycin sensitivity

Summary Forty lymphoblastoid (lymphoid) lines were established from 42 volunteer blood donors, including healthy individuals and patients with head and neck carcinomas. Each peripheral blood sample was split into two portions, one for the establishment of a lymphoid line and the other for short-term culture, which was used to estimate bleomycin sensitivity by cytogenetic procedures. Twenty lymphoid lines were selected at random to compare bleomycin sensitivity with data obtained from short-term lymphocyte cultures. In each set, bleomycin sensitivity of lymphoid cells was similar to that of the lymphocytes. The lymphoid lines, which can be propagated for an unlimited supply of relatively homogeneous cellular material, will be useful for a variety of future investigations. This investigation was supported by grants from the John S. Dunn Foundation, Houston, TX, the Esther Knispel Fund administered by The University of Texas M. D. Anderson Cancer Center, Houston, TX, and Department of Health and Human Services PHS grant DE 07007.     

Isolation of a gene involved in 1,3-beta-glucan synthesis in Aspergillus nidulans and purification of the corresponding protein.

Saccharomyces cerevisiae has two highly homologous genes, FKS1 and FKS2, which encode interchangeable putative catalytic subunits of 1,3-beta-glucan synthase (GS), an enzyme that synthesizes an essential polymer of the fungal cell wall. To determine if GS in Aspergillus species is similar, an FKS homolog, fksA, was cloned from Aspergillus nidulans by cross-hybridization, and the corresponding protein was purified. Sequence analysis revealed a 5,716-nucleotide coding region interrupted by two 56-bp introns. The fksA gene encodes a predicted peptide of 229 kDa, FksAp, that shows a remarkable degree of conservation in size, charge, amino acid identity, and predicted membrane topology with the S. cerevisiae FKS proteins (Fksps). FksAp exhibits 64 and 65% identity to Fks1p and Fks2p, respectively, and 79% similarity. Hydropathy analysis of FksAp suggests an integral membrane protein with 16 transmembrane helices that coincide with the transmembrane helices of the Saccharomyces Fksps. The sizes of the nontransmembrane domains are strikingly similar to those of Fks1p. The region of FksAp most homologous to the Saccharomyces FKS polypeptides is a large hydrophilic domain of 578 amino acids that is predicted to be cytoplasmic. This domain is 86% identical to the corresponding region of Fks1p and is a good candidate for the location of the catalytic site. Antibodies raised against a peptide derived from the FksAp sequence recognize a protein of approximately 200 kDa in crude membranes and detergent-solubilized active extracts. This protein is enriched approximately 300-fold in GS purified by product entrapment. Purified anti-FksAp immunoglobulin G immunodepletes nearly all of the GS activity in crude or purified extracts when Staphylococcus aureus cells are used to precipitate the antibodies, although it does not inhibit enzymatic activity when added to extracts. The purified GS is inhibited by echinocandins with a sensitivity equal to that displayed by whole cells. Thus, the product of fksA is important for the activity of highly purified preparations of GS, either as the catalytic subunit itself or as an associated copurifying subunit that mediates susceptibility of enzymatic activity to echinocandin inhibition.     

The UL2 Open Reading Frame of Bovine Herpesvirus 1 Encodes a Uracil-DNA Glycosylase

Sequence analysis within the unique long segment of the bovine herpesvirus 1 (BHV-1) genome previously identified an open reading frame (ORF), designated UL2, whose deduced polypeptide of 204 amino acids contained a consensus uracil-DNA glycosylase (UDGase) signature sequence. To determine whether the BHV-1 UL2 ORF product has UDGase activity, we positioned the UL2 sequence downstream of the T7 promoter on the vector pET-28b(+) and expressed it in Escherichia coli. Upon induction with isopropyl β-D -thiogalactopyranoside these cells produced a 23-kDa protein, the molecular mass of which was in accordance with the prediction from the nucleotide sequence. A one-step purification procedure using nickel-chelating affinity chromatography resulted in a homogeneous preparation of this protein, which displayed specific UDGase activity in an in vitro enzyme assay. These results provide evidence that the BHV-1 UL2 gene does encode a UDGase.     

Virus-encoded superantigens.

Superantigens are microbial agents that have a strong effect on the immune response of the host. Their initial target is the T lymphocyte, but a whole cascade of immunological reactions ensues. It is thought that the microbe engages the immune system of the host to its own advantage, to facilitate persistent infection and/or transmission. In this review, we discuss in detail the structure and function of the superantigen encoded by the murine mammary tumor virus, a B-type retrovirus which is the causative agent of mammary carcinoma. We will also outline what has more recently become known about superantigen activity associated with two human herpesviruses, cytomegalovirus and Epstein-Barr virus. It is likely that we have only uncovered the tip of the iceberg in our discovery of microbial superantigens, and we predict a flood of new information on this topic shortly.     

Analysis of red blood cell motion through cylindrical micropores: effects of cell properties.

Filtration through micropores is frequently used to assess red blood cell deformability, but the dependence of pore transit time on cell properties is not well understood. A theoretical model is used to simulate red cell motion through cylindrical micropores with diameters of 3.6, 5, and 6.3 microns, and 11-microns length, at driving pressures of 100-1000 dyn/cm2. Cells are assumed to have axial symmetry and to conserve surface area during deformation. Effects of membrane shear viscosity and elasticity are included, but bending resistance is neglected. A time-dependent lubrication equation describing the motion of the suspending fluid is solved, together with the equations for membrane equilibrium, using a finite difference method. Predicted transit times are consistent with previous experimental observations. Time taken for cells to enter pores represents more than one-half of the transit time. Predicted transit time increases with increasing membrane viscosity and with increasing cell volume. It is relatively insensitive to changes in internal viscosity and to changes in membrane elasticity except in the narrowest pores at low driving pressures. Elevating suspending medium viscosity does not increase sensitivity of transit time to membrane properties. Thus filterability of red cells is sensitively dependent on their resistance to transient deformations, which may be a key determinant of resistance to blood flow in the microcirculation.