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K Cunningham

Publications and source records attributed to K Cunningham.

At least 37 records · Page 2Linked to original sources
PubMed1989

SecA protein, a peripheral protein of the Escherichia coli plasma membrane, is essential for the functional binding and translocation of proOmpA.

We have reconstituted protein translocation across plasma membrane vesicles of Escherichia coli using purified proOmpA and trigger factor, a 63 kd soluble protein. Treatment of membrane vesicles with urea inactivates them for translocation unless a factor present in cytoplasmic extracts is added during the translocation reaction. Sedimentation analysis showed that the stimulatory activity is of distinctly higher mol. wt than trigger factor. Cytoplasmic extracts from a strain that greatly overproduces the SecA protein are highly enriched in the stimulatory activity for untreated membranes and restore translocation to urea-treated membranes, suggesting that this protein is the stimulatory factor. This assay was used to monitor the isolation of SecA protein from the overproducing strain. The purified protein is soluble, yet binds peripherally to membranes with high affinity and supports translocation. Using pure proOmpA, SecA protein, trigger factor and urea-treated membranes, the protein export process was resolved into binding and translocation steps. We find that proOmpA binds to membrane vesicles with or without SecA protein, but that translocation only occurs when SecA was bound prior to proOmpA.

Amino Acid Isomerases↗
PubMed1989

SecA protein hydrolyzes ATP and is an essential component of the protein translocation ATPase of Escherichia coli.

Bacterial protein export requires two forms of energy input, ATP and the membrane electrochemical potential. Using an in vitro reaction reconstituted with purified soluble and peripheral membrane components, we can now directly measure the translocation-coupled hydrolysis of ATP. This translocation ATPase requires inner membrane vesicles, SecA protein and translocation-competent proOmpA. The stimulatory activity of membrane vesicles can be blocked by either antibody to the SecY protein or by preparing the membranes from a secY-thermosensitive strain which had been incubated at the non-permissive temperature in vivo. The SecA protein itself has more than one ATP binding site. 8-azido-ATP inactivates SecA for proOmpA translocation and for translocation ATPase, yet does not inhibit a low level of ATP hydrolysis inherent in the isolated SecA protein. These data show that the SecA protein has a central role in coupling the hydrolysis of ATP to the transfer of pre-secretory proteins across the membrane.

Adenosine Triphosphatases↗
PubMed1989

Specific recognition of the leader region of precursor proteins is required for the activation of translocation ATPase of Escherichia coli.

The ATP-hydrolytic activity of SecA protein is stimulated up to 100-fold by the translocation-competent precursor to outer membrane protein A (pro-OmpA) in conjunction with inner-membrane vesicles bearing active SecY [Lill, R., Cunningham, K., Brundage, L., Ito, K., Oliver, D. & Wickner, W. (1989) EMBO J. 8, 961-966]. This reaction is saturable, with Michaelis-Menten kinetics for an enzyme with two substrates, ATP and pro-OmpA, and is defined as translocation ATPase. Another precursor protein, pre-PhoE, is also a substrate for this translocation ATPase. Neither OmpA nor its synthetic leader peptide are effective substrates for translocation ATPase, suggesting that both domains of the complete precursor are necessary for the reaction. The leader peptide is a potent inhibitor and apparently competes with pro-OmpA for necessary binding sites on translocation ATPase. After a brief preincubation, the activity of translocation ATPase becomes resistant to inhibition by leader peptide, suggesting that the leader peptide is recognized at an early step in the protein translocation pathway. Our enzymological studies show that translocation ATPase recognizes and functionally binds the leader region of precursor proteins.

Amino Acid Sequence↗
PubMed1989

Detergent disruption of bacterial inner membranes and recovery of protein translocation activity.

Isolation of the integral membrane components of protein translocation requires methods for fractionation and functional reconstitution. We treated inner-membrane vesicles of Escherichia coli with mixtures of octyl beta-D-glucoside, phospholipids, and an integral membrane carrier protein under conditions that extract most of the membrane proteins into micellar solution. Upon dialysis, proteoliposomes were reconstituted that supported translocation of radiochemically pure [35S]pro-OmpA (the precursor of outer membrane protein A). Translocation into these proteoliposomes required ATP hydrolysis and membrane proteins, indicating that the reaction is that of the inner membrane. The suspension of membranes in detergent was separated into supernatant and pellet fractions by ultracentrifugation. After reconstitution, translocation activity was observed in both fractions, but processing by leader peptidase of translocated pro-OmpA to OmpA was not detectable in the reconstituted pellet fraction. Processing activity was restored by addition of pure leader peptidase as long as this enzyme was added before detergent removal, indicating that the translocation activity is not associated with detergent-resistant membrane vesicles. These results show that protein translocation activity can be recovered from detergent-disrupted membrane vesicles, providing a first step towards the goal of isolating the solubilized components.

Bacterial Outer Membrane Proteins↗
PubMed1986

Lack of communication between LC2 light chain and the SH1 region of myosin S-1 studied by 19F-NMR.

Myosin subfragment-1 (S-1) which contains the LC2 light chain has been labelled with fluorine to allow an 19F-NMR study of the coupling and energetics of structural changes in the myosin head. Two fluorine-containing reagents, N-4-(trifluoromethyl)phenyl iodoacetamide and N-3,5-di(trifluoromethyl)phenyl iodoacetamide, have been used to label the myosin heavy chain at the unusually reactive sulfhydryl-1 (SH1) position. The chemical shift of both reagents on S-1 is sensitive to a structural transition in the region of SH1 which occurs upon increasing the temperature from 0 degrees C to 35 degrees C. The midpoint of the transition in both papain and chymotryptic S-1 is at approximately 11 degrees C at pH 7 (0.1 M CKl). The temperature dependence of the chemical shift may be fit assuming a two-state equilibrium where delta G degree' (T) = 101-110T +0.386 T2 (where T is the temperature in Kelvin). Both delta H degree' (T) and delta S degree' (T) have a small temperature dependence from 0 to 35 degrees C: at 20 degrees C, delta H degree' (T) = -33 kcal/mol. delta S degree' (T) = -116 e.u. and delta Cp = -226 cal/mol per deg (pH 7.0, 0.1 M KCl). The NMR data indicate that the presence of the LC2 light chain in papain S-1 does not modify the structure of S-1 in the vicinity of SH1, nor does it modify the energetics of the structural transition from that seen in its absence with chymotryptic S-1. The presence of calcium which is bound by the LC2 of papain S-1 also does not alter the energetics of the transition. Thus it would appear that the LC2 light chain (on myosin S-1) does not participate in the two-state transition, nor does it interact strongly with regions of the heavy chain which participate in the transition.

Adenosine Triphosphatases↗
PubMed1986

Colour and brightness coding in the central nervous system: theoretical aspects and visual evoked potentials to homogeneous red and green stimuli.

We designed visual evoked potentials experiments to study the differential aspects of colour and brightness coding in man. The substitution of equally bright red and green stimuli for a background yellow was investigated and compared with different luminance increments and decrements of red and green. A dominant N87 component was found for a colour change from yellow to brighter red colours, which was less pronounced for green and absent for yellow luminance changes. It is also absent for pure red luminance increments and green luminance changes, but reappears with red luminance decrements or red-offset. The data are discussed within the framework of a new concept of how the visual system fuses red-green information and black-white border information. Retinal X-cells can transmit colour and high spatial frequency achromatic information simultaneously by encoding only the presence of edges (a.c.) for the black-white stimuli and the presence of both edges (a.c.) and uniform areas of colour (d.c.) for red-green stimuli. Phylogenetically this kind of information transmission enables colour vision to be implemented in a retina such as the cat's by adding only a second class of cones. Barlow's economy principle will be violated for colour in the periphery, but restored early in the striate cortex where there is an early decoding of the combined chromatic and achromatic information by the concentric double opponent cells. The N87 behaviour correlates with the proposed discharge of peripheral X-type cells, but not with the discharge of cortical double opponent concentric or simple cells, which no longer respond to homogeneous colour stimuli. It is suggested that N87 may be generated by geniculate afferents in the dendritic arborization of cortical cells, reflecting the behaviour of peripheral units, and thus the violation of the economy principle, rather than the next step in cortical processing. The early cortical restoration of the economy principle is supported by the absence of any further dissociated behaviour for colour and brightness in later components.

Adult↗
PubMed1986

A Thy-1- mutant defining a gene acting in trans position to regulate cell-surface Thy-1 glycoprotein expression and Thy-1 messenger RNA content.

The Thy-1 glycoprotein is a differentiation antigen which exhibits tissue-specific regulation. A mutant of a Thy-1.1+ T-cell lymphoma has been isolated which does not express Thy-1 glycoprotein on the cell surface and does not accumulate Thy-1 mRNA in the cytoplasm. Hybrids between the mutant and a Thy-1.2+ T-cell lymphoma express 20-30-fold lower levels of Thy-1 glycoprotein on their cell surface compared to wild-type T-cell lymphomas, and they have correspondingly low levels of cytoplasmic Thy-1 mRNA. A revertant of one hybrid was isolated which expressed wild-type levels of both Thy-1 alleles on its surface and contained correspondingly increased levels of Thy-1 mRNA. A Thy-1+ revertant of the Thy-1- mutant was isolated by cell sorting. A second generation Thy-1- mutant could be isolated from this revertant which also did not accumulate Thy-1 mRNA and which behaved in a way similar to the first generation mutant when hybridized to a Thy-1.2+ lymphoma. No changes in the structure or copy number of the Thy-1 structural gene could be detected in this series of mutants and revertants. These properties are consistent with a mutation in one (or more) gene(s) which acts in trans position to regulate Thy-1 glycoprotein expression.

Animals↗
PubMed1986

Simulation of 'stationary' SAP and SEP phenomena by 2-dimensional potential field modelling.

In order to model the distribution of potentials in the hand due to antidromic SAP propagation and in the body due to afferent conduction of the median nerve volley, 2-dimensional matrices of the appropriate shape were constructed, each containing a 'generator' consisting of up to 3 'source' and 3 'sink' points. The value of the field potential at other sites was calculated using a finite difference method. It was shown that the potential gradient is virtually zero in matrix zones which are separated from the region containing the generator by a constriction in the boundary of the conductor. Points on the far side of the constriction remain virtually equipotential, at a level determined by the potential at the junction. This is naturally influenced by the proximity of the generator, so that as the generator approaches the constriction a potential difference will develop between points on the far side, irrespective of their distance from the junction, and other remote parts of the matrix. In the context of human SAPs and SEPs, such factors may be of paramount importance in the generation of so-called 'stationary' or 'far-field' potentials. With additional postulates concerning the manner in which the SAP is attenuated by the termination of axons as it propagates through the hand, and the course taken by the median nerve volley between the arm and neck, it was possible to model the majority of stationary SAP phenomena described by Kimura et al. (1984), and also the distribution and latency of the P9 SEP component following median nerve stimulation.

Action Potentials↗
PubMed1985

Occipital distribution of foveal half-field responses.

Foveal left and right half-field responses were recorded in six subjects (12 half-fields) and compared with wide half-field responses. In the foveal responses a general trend was seen for the negative-positive-negative (NPN) complex to extend to the contralateral channels, replacing the contralateral positive-negative-positive (PNP) complex found with wide half-field stimulation. Many individual variations in foveal response morphology were seen, which may be understood in terms of anatomical variation. Individual differences between foveal half-fields in the same subject may be as great as those between subjects.

Evoked Potentials, Visual↗
PubMed1984

Regulation of Thy-1 and Pgp-1 glycoproteins in hybrids between T-cell lymphomas and Abelson-leukemia-virus-induced lymphomas.

Most cells in the normal adult mouse thymus express Thy-1 glycoprotein but do not express Pgp-1 glycoprotein. In contrast, cells of the mouse B-cell lineage are Thy-1 negative and Pgp-1 positive. Somatic cell hybrids between pseudodiploid Thy-1+, Pgp-1- T-cell lymphomas and pseudotetraploid Thy-1-, Pgp-1+ Abelson-leukemia-virus-induced cell lines do not express detectable cell-surface Thy-1 but show activation of the T-cell Pgp-1 glycoprotein. Hybrids between pseudodiploid lines, in contrast, show extinction of Pgp-1. Thy-1+ or Pgp-1+ revertants were isolated by cell sorting from hybrids in which extinction occurred, demonstrating that all genes required for expression of these cell-surface antigens were present in antigen-negative hybrids. Thy-1- hybrids did not contain detectable cytoplasmic Thy-1 messenger RNA, while Thy-1 message could be detected in parental lines and Thy-1+ revertants. No obvious rearrangements of the Thy-1 structural genes could be demonstrated in Thy-1- hybrids and their Thy-1+ revertants, nor could rearrangements be demonstrated when parental cells and Thy-1- hybrids were compared. These results are consistent with the idea that diffusible gene products regulate both Thy-1 and Pgp-1 expression in these hybrids. These products act in a gene dosage-dependent manner in somatic cell hybrids. Regulation of Thy-1 is at the level of either messenger RNA transcription or processing.

Abelson murine leukemia virus↗
PubMed1984

Colour and brightness components of foveal visual evoked potentials in man.

The different effects of colour and brightness on the transient visual evoked potentials to a foveal stimulus have been investigated in a psycho-physically controlled stimulus set-up, in which equally bright red, orange and green stimuli were substituted for a standard yellow stimulus. These colour-evoked responses were compared with the responses to additional brightness increments and decrements of each of the colour stimuli. An initial component of small amplitude, P55, was followed by a colour-dominated component, N87, and a luminance-dominated component, P120, with a maximum at the occipital electrode. Both N87 and P120 showed a decline in amplitude at the parietal electrode and P120 had a reversed polarity at FZ. These results indicate that the responses to equally bright green and red stimuli have closely similar wave forms, but that this changes rapidly with additional brightness differences. Comparison with the reports of subdural recording of colour-evoked potentials in the macaque striate cortex suggests that P55 corresponds with the primary excitation via geniculo-cortical fibres and that N87 and P120 represent later stages of cortical processing.

Color Perception↗
PubMed1984

Expression of the Thy-1 glycoprotein gene by DNA-mediated gene transfer.

We isolated a gene encoding the Thy-1.2 glycoprotein from a recombinant library constructed from BALB/c mouse DNA. To evaluate the expression of this cloned gene in different genomic environments, we introduced it into cell lines derived from fibroblast, lymphoid, and neuronal tissues by DNA-mediated gene transfer. When integrated into the genome of mouse L cells, cell-surface Thy-1 can be detected with anti-Thy-1 monoclonal antibodies. These L-cell lines contain between two and four copies of the cloned Thy-1 gene stably integrated in the host genome. After subcloning into a plasmid vector containing the bacterial Eco-gpt gene as a selectable marker, the Thy-1 gene was introduced into the Thy-1-deficient mouse lymphoma AKR1 (Thy-1-d), and the rat neuronal cell line, B50. The resulting transformants also contain two to four copies of the cloned Thy-1 gene but express up to 50-fold more cell-surface Thy-1.2 than the L cell transformants. The expression of vastly differing amounts of cell-surface Thy-1 from similar numbers of genes suggests that the gene encoding this differentiation antigen is under tissue-specific regulation.

Animals↗
PubMed1984

Donor-specific antibodies in renal allograft recipients. A reevaluation of the 51Cr-release assays comparing fibroblast and lymphoid target cells.

Donor-specific alloantibody was monitored in recipients of 81 renal allografts by 51Cr release assays. Complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity were both followed. In addition to the conventional lymphoid target cells nonlymphoid target cells, namely fibroblasts from the donor, were used. Although antibody against donor lymphoid cells was found more frequently, associations with graft loss and with rejection were greater with antibody against fibroblasts. The superior association of antibody against fibroblasts with clinical events suggests that wider use of nonlymphoid target cells would be of value in studies of alloantibody responses in renal transplant recipients.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1981

Structure of the Tetrahymena pyriformis rRNA gene. Nucleotide sequence of the transcription termination region.

The 3'-terminal regions of the 25 S rRNA and 35 S rRNA were mapped in the HindIIIC fragment of the T. pyriformis rRNA gene (Niles, E. G., and Jain, R. K. (1981) Biochemistry 20, 905-909). The precise locations of these termini were identified by S1 nuclease mapping. The 35 S rRNA was shown to contain two major 3'-ends, one identical to the 3'-end of the mature 25 S rRNA and one which is 15 bases longer. The nucleotide sequence of 440 base pairs of DNA in the terminator region was determined. There are several oligo T stretches and regions of dyad symmetry. Three models of transcription termination are presented which are based on possible secondary structures in the 3'-terminal region of the 35 S rRNA.

Animals↗
PubMed1981

Effect of gene dosage on cell-surface expression of Thy-1 antigen in somatic cell hybrids between Thy-1- Abelson-leukemia-virus induced lymphomas and Thy-1+ mouse lymphomas.

Hybrids between pseudodiploid Thy-1.1+ lymphomas and Thy 1.2- pseudodiploid Abelson-leukemia-virus-induced-(ALV-induced) lymphomas express Thy-1 glycoprotein on their cell surface. These Thy-1+ hybrids invariably express the Thy 1.1 allelic form of the glycoprotein and may be either Thy 1.2+ or Thy 1.2-. Sublines expressing both Thy 1.1 and Thy 1.2 can be isolated from Thy 1.1+, Thy 1.2- hybrids by cell sorting. In contrast to hybrids with pseudodiploid ALV-induced lymphomas, hybrids between Thy 1.1+ lymphomas and pseudotetraploid Thy 1.2- Abelson-leukemia-virus-induced lymphomas do not express Thy-1 glycoprotein on their cell surface and Thy-1 glycoprotein cannot be detected in detergent extracts of these cells. Thy-1+ revertants were isolated from one of the Thy-1- hybrids by cell sorting. - These results demonstrate a gene dosage effect for the expression of the Thy-1 glycoprotein in somatic cell hybrids. They are consistent with the idea that diffusable gene products regulate Thy-1-glycoprotein expression in these hybrids. They also suggest that there may be additional, apparently cis-active, regulatory mechanisms which determine the ability of the Thy-1 structural genes of the Abelson-leukemia-virus-induced lymphoma parent to be expressed in somatic cell hybrids.

Abelson murine leukemia virus↗
PubMed1981

Cor pulmonale in cystic fibrosis.

A VCG and an orthogonal ECG were done on 66 ambulatory patients with cystic fibrosis. Arteriolized pO2, FEV1/VC and MBC were related to electrocardiographic measurements. In this group of adolescent and young adult patients the most helpful electrocardiographic sign of cor pulmonale is a low voltage representing the left ventricle. The four best indicators from the cardiogram are (1) Rx less than .8 mv; (2) Rx/Sx less than 4.0; (3) Tx less than .4 mv; and (4) MLVF + MLVH less than 2.5 mv. If more than one indicator is found cor pulmonale is likely present. Increased voltage over the right ventricle does not identify patients with cor pulmonale.

Adolescent↗
PubMed1980

Cell surface molecules of Friend erythroleukemias: decrease in T200 glycoprotein expression after induction.

Monoclonal antibodies against the Thy-1 and T200 glycoproteins were used to study the expression of cell surface molecules on mouse hematopoietic cell lines. Friend erythroleukemias express T200 glycoprotein but do not express significant amounts of Thy-1 glycoprotein on their cell surface. The rate of T200 glycoprotein synthesis in maximally-induced Friend erythroleukemia 745.6 cells is less than 10% that in noninduced cells, although total protein synthesis shows only a twofold decline and induced cells express 2-6-fold less T200 glycoprotein on their surface compared to noninduced cells. T200 glycoprotein expression is reduced in a variant cell line obtained by selection for growth in dimethylsulfoxide, showing that the reduction in T200 glycoprotein synthesis characteristic of induced cells is an event that can be dissociated from commitment and hemoglobin synthesis. Analysis of T200 glycoprotein negative cell lines, isolated by cytotoxic immunoselection against T200 glycoprotein, indicates that the presence of T200 glycoprotein on the cell surface is not necessary for induction of hemoglobin synthesis and terminal differentiation of Friend erythroleukemias.

Animals↗