Type II protein secretion by Pseudomonas aeruginosa: genetic suppression of a conditional mutation in the pilin-like component XcpT by the cytoplasmic component XcpR

Pseudomonas aeruginosa exports a number of hydrolytic enzymes and toxins using the type II or general secretion pathway, found in a variety of Gram-negative bacteria and requiring the functions of at least 12 gene products (XcpP–Z and PilD/XcpA in P. aeruginosa ). A number of these gene products are homologues of components of the type IV pilus biogenesis system, including four proteins, XcpT–W, which are highly similar to the pilin subunit in their size, localization and post-translational modifications. These proteins, in addition to the pilin subunit, are cleaved and methylated by the PilD/XcpA prepilin peptidase, but their interactions with other components of the export apparatus are unclear. Using a medium developed for the selection of export-proficient P. aeruginosa strains, we have isolated temperature-sensitive mutations in the xcpT gene and extragenic suppressors for one of the mutants. These suppressors fall into two classes, one that maps outside of the xcpP–Z gene cluster and may define additional cellular functions that are required for export, and a second that maps to the xcpR gene product and indicates a potential protein–protein interaction connecting two different cellular compartments and required for the assembly or function of the export apparatus.     

Structure of a pleiomeric form of poly d(AT):poly d(AT)

A chemically simple polynucleotide duplex, poly d(AT):poly d(AT), has been trapped in a fibrous form with a complex helical secondary structure with a large (7.4 nm) axial repeat 24 nucleotides long. The motif which is repeated by the symmetry elements is a hexanucleotide in which two residues (both TpA) have the less common gauche minus conformation at C3'-O3' and consequently distinctive phosphate orientations. This reinforces earlier conclusions that PypPu nucleotides tend to have different shapes from PupPy nucleotides and that DNA surfaces may signal what base sequences lie beneath them. The morphological differences between this pleiomeric DNA polymer and closely-related, but more symmetrical allomorphs are just as great as those observed in short DNA fragments in crystals.     

Synemin and vimentin are components of intermediate filaments in avian erythrocytes

Synemin, a high-molecular-weight protein associated with intermediate filaments in muscle, and vimentin, an intermediate-filament subunit found in many different cell types, have been identified by immunologic and electrophoretic criteria as components of intermediate filaments in mature avian erythrocytes. Desmin, the predominant subunit of intermediate filaments in muscle, has not been detected in these cells. Two dimensional immunoautoradiography of proteolytic fragments of synemin and vimentin demonstates that the erythrocyte proteins are highly homologous, if not identical, to their muscle counterparts. Double immunoflurorescence reaveals that erythrocyte synemin and vimentin co-localize in a cytoplasmic network of sinuous filaments that extends from the nucleus to the plasma membrane and resists aggregation by colcemid. Erythrocytes that are attached to glass cover slips can be sonicated to remove nuclei and nonadherent regions of the plasma membrane; this leaves elliptical patches of adherent membrane that retain mats of vimentin- and synemin-containing intermediate filaments, as seen by immunofluorescence and rotary shadowing. Similarly, mechanical enucleation of erythrocyte ghosts in suspension allows isolation of plasma membranes that retain a significant fraction of the synemin and vimentin, as assayed by electrophoresis, and intermediate filaments, as seen in thin sections. Both synemin and vimentin remain insoluble along with spectrin and actin, in solutions containing nonionic detergent and high salt. However, brief exposure of isolated membrane to distilled water releases the synemin and vimentin together in nearly pure form, before the release of significant amounts of spectrin and actin. These data suggest that avian erythrocyte intermeditate filaments are somehow anchored to the plasma membrane; erythrocytes may thus provide a simple system for the study of intermediate filaments and their mode of interaction with membranes. In addition, these data, in conjunction with previous data from muscle, indicate that synemin is capable of associating with either desmin or vimentin and may thus perform a special role in the structure or function of intermediate filaments in erythrocytes as well as muscle.     

A synthetic strand of cardiac muscle: its passive electrical properties

The passive electrical properties of synthetic strands of cardiac muscle, grown in tissue culture, were studied using two intracellular microelectrodes: one to inject a rectangular pulse of current and the other to record the resultant displacement of membrane potential at various distances from the current source. In all preparations, the potential displacement, instead of approaching a steady value as would be expected for a cell with constant electrical properties, increased slowly with time throughout the current step. In such circumstances, the specific electrical constants for the membrane and cytoplasm must not be obtained by applying the usual methods, which are based on the analytical solution of the partial differential equation describing a one-dimensional cell with constant electrical properties. A satisfactory fit of the potential waveforms was, however, obtained with numerical solutions of a modified form of this equation in which the membrane resistance increased linearly with time. Best fits of the waveforms from 12 preparations gave the following values for the membrane resistance times unit length, membrane capacitance per unit length, and for the myoplasmic resistance: 1.22 plus or minus 0.13 x 10-5 omegacm, 0.224 plus or minus 0.023 uF with cm-minus 1, and 1.37 plus or minus 0.13 x 10-7 omegacm-minus 1, respectively. The value of membrane capacitance per unit length was close to that obtained from the time constant of the foot of the action potential and was in keeping with the generally satisfactory fit of the recorded waveforms with solutions of the cable equation in which the membrane impedance is that of a single capacitor and resistor in parallel. The area of membrane per unit length and the cross-sectional area of myoplasm at any given length of the preparation were determined from light and composite electron micrographs, and these were used to calculate the following values for the specific electrical membrane resistance, membrane capacitance, and the resistivity of the cytoplasm: 20.5 plus or minus 3.0 x 10-3 omegacm-2, l.54 plus or minus 0.24 uFWITHcm-minus 2, and 180 plus or minus 34 omegacm, respectively.     

Interleukin 2-mediated induction of lytic activity in a cloned murine CTL line

We have analyzed the ability of interleukin 2 (IL 2) to induce lytic activity within a cloned murine H-2Dd-specific CTL line. Weakly lytic CTL harvested 6 to 7 days after previous stimulation with irradiated DBA/2J spleen cells and conditioned medium from secondary MLC (MLC SN) could be reactivated to high antigen-specific lytic activity with highly purified gibbon IL 2 or E. coli-produced human recombinant DNA IL 2. Dose-response curves with IL 2 and MLC SN suggest that IL 2 may be the principal detectable activity in MLC SN that is active on these CTL. Doses of IL 2 or MLC SN that were saturating for the induction of lytic activity were suboptimal for the expression of DNA synthesis measured by 3HTdR incorporation. This is consistent with a mechanism in which different threshold IL 2 concentrations are required to induce these two biologic responses. Finally, we show that IFN-gamma has little effect on the expression of lytic activity either alone or in combination with IL 2 in this bioassay.     

Circular dichroism evidence for G·U and G·T base pairing in poly[r(G-U)] and poly[d(G-T)]

We have measured the circular dichroism (CD) and absorption properties of poly[r(G-U)] and poly [d(G-T)] over a wide range of Na⁺ concentrations and temperatures. We find evidence for self-complexed forms of these polymers at lower temperatures and/or higher Na⁺ concentrations than generally needed for double-strand formation in other DNA and RNA polymers. These self-complexes could be composed of double-stranded regions with weak G·U or G·T base pairs.     

A.T and C.C+ base pairs can form simultaneously in a novel multistranded DNA complex

Previous experiments have established that in certain synthetic oligomeric DNA sequences, including mixtures of d(AACC)5 with d(CCTT)5, adenine-thymine (A.T) base pairs form to the exclusion of neighboring protonated cytosine-cytosine (C.C+) base pairs [Edwards, E., Ratliff, R., & Gray, D. (1988) Biochemistry 27, 5166-5174]. In the present work, circular dichroism and other measurements were used to study DNA oligomers that represented two additional classes with respect to the formation of A.T and/or C.C+ base pairs. (1) One class included two sets of repeating pentameric DNA sequences, d(CCAAT)3-6 and d(AATCC)4,5. For both of these sets of oligomers, an increase in the magnitude of the long-wavelength positive CD band centered at about 280 nm occurred as the pH was lowered from 7 to 5 at 0.1 and 0.5 M Na+, indicating that C.C+ base pairs formed. Even though it may have been possible for these oligomers to form duplexes with two antiparallel A.T base pairs per pentamer, no A.T base pairing was detected by monitoring the CD changes at 250 nm. Thus, spectral data showed that as few as 40% C.C+ base pairs were stable in two sets of oligomers in which A.T base pairs did not form adjacent to, or in place of, C.C+ base pairs. (2) Another class of oligomer was represented by d(C4A4T4C4), which was studied by CD, HPLC, and centrifugation experiments. We confirmed previous work that this sequence was able to form both types of base pairs as the pH and temperature were lowered [Gray, D., Cui, T., & Ratliff, R. (1984) Nucleic Acids Res. 12, 7565-7580].(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of carrageenan on spontaneous and antibody-dependent cell-mediated cytotoxicity

We examined the effect of carrageenan on in vitro antibody-dependent cell-mediated cytolysis (ADCC) and spontaneous cell-mediated cytolysis (SCMC) in cultures of human peripheral blood mononuclear cells (PBL). Carrageenan, when present in the assay, nonspecifically reduced ADCC and SCMC against both Chang and chicken erythrocyte (CRBC) target cells. This reduction in cytotoxicity could not be attributed entirely to the macrophage toxic and complement-inhibitory properties of carrageenan because neither removal of complement nor macrophage depletion prevented the dose-dependent inhibition. In contrast, pretreatment of effector PBL, with carrageenan followed by removal of Carrageenan by washing did not alter ADCC or SCMC against Chang cells, which are mediated by nonphagocytic cells, but reduced both ADCC and SCMC activity against CRBC targets, which are mediated in part by macrophages. Thus, Carrageenan, when present in in vitro cell-mediated cytotoxicity assays, causes a nonspecific impairment of cytotoxicity that is independent of its anticomplement or macrophage-toxic properties.