The synthesis of polyamide-oligonucleotide conjugate molecules.

We have developed methods for the synthesis of peptide-oligodeoxyribonucleotide conjugate molecules in particular, and polyamide-oligonucleotide conjugates in general. Synthesis is carried out by a solid-phase procedure and involves the assembly of a polyamide on the solid support, conversion of the terminal amino group to a protected primary aliphatic hydroxy group by reaction with alpha, omega-hydroxycarboxylic acid derivatives, and finally oligonucleotide synthesis using phosphoramidite chemistry. The conjugate molecules can be used as DNA probes, with the polyamide component carrying one or more non-radioactive markers. These conjugates also have the potential to be used as anti-sense inhibitors of gene expression, with the peptide segment acting as a targeting moiety.     

Expression of the Chlamydia trachomatis major outer membrane protein-encoding gene in Escherichia coli: role of the 3' end in mRNA stability

The major outer membrane protein (MOMP)-encoding gene (omp1) of Chlamydia trachomatis has been cloned into Escherichia coli and partially sequenced. This recombinant gene expresses a full-length 40-kDa product, which is recognized by a monoclonal antibody directed against the species-specific epitope of MOMP. The recombinant omp1 is expressed in either insertion orientation, indicating that it utilizes its own promoter system. The endogenous omp1 promoter possesses a relatively low activity despite the high level of MOMP expression. Deletion of a 520-bp fragment at the 3' end encoding 39 amino acids (aa) at the C terminus and the remainder of the noncoding region leads to a significant decrease in mRNA stability and loss of protein synthesis. When the MOMP-encoding plasmid was introduced into E. coli minicells, it expressed 40- and 43-kDa proteins; however, inhibition of post-translational processing by ethanol revealed only a 43-kDa protein. These data indicate that the unprocessed omp1 gene product contains a 22-aa leader sequence which is cleaved during translocation to the outer membrane, to yield a processed 40-kDa protein. The recombinant MOMP was localized to the outer membrane E. coli fraction, comparable to the location of the native C. trachomatis protein.     

The perception of painful and nonpainful stimuli during voluntary motor activity in man

Previous studies have shown that voluntary movement diminishes the transmission of cutaneous afferent input through the dorsal column-medial lemniscal system, and also raises the threshold for detecting nonpainful, cutaneous stimuli (electrical shocks). Although there is some evidence that pain elicited by electrical stimulation is diminished during movement, no studies have tested the effect of movement on the perception of pain produced by natural stimulation. For this reason, we tested the effects of voluntary motor activity on the perception of noxious thermal stimuli in human volunteers. We first developed a motor paradigm in which the thermal stimulation could be applied to the immobile limb (isometric elbow flexion-extension). Both isometric and isotonic muscle contractions about the elbow increased the threshold for detecting weak cutaneous stimuli (electrical shocks) applied to the forearm, and to a lesser extent the detection of stimuli applied to the dorsum of the hand. Afterwards, noxious and innocuous heat stimuli were applied to the forearm during isometric contractions and at rest. Magnitude estimates for the intensity of the pain, as well as latency measures of the onset of pain, were recorded. We found no evidence that isometric motor activity diminished either the threshold for pain or the subjective intensity of the noxious and innocuous thermal stimuli. Thus, motor activity decreases the ability to detect weak low-threshold cutaneous inputs, but has no effect on the perception of warmth and heat pain.     

The role of phospholipase A2 in calcium-induced damage in cardiac and skeletal muscle

Summary This study compares the action of inhibitors of the eicosanoid cascade on calcium-induced myofilament damage in cardiac muscle of the perfused frog heart and incubated frog ventricle slices, and in skeletal muscle of incubated mammalian diaphragm and isolated and saponin-skinned amphibian pectoris cutaneous muscle. Mepacrine (10^-5M) and indomethacin (3×10^-6M) protected completely against myofilament damage induced by entry of calcium in the calcium-paradox in frog heart. However, inhibition of phospholipase A₂ (PLA₂) (with chlorpromazine, 2×10^-4M, or mepacrine, 10^-5M, 5x10^-5M), of cyclo-oxygenase enzymes (with indomethacin, 3x10^-6M to 10^-5M or BW755C, 3.8x10^-4M), or of lipoxygenase enzymes (with BW755C, 3.8x10^-4M or nordihydroguaiaretic acid, 2x10^-6M or 5x10^-6M) all failed in intact cardiac or skeletal muscle cells to prevent the myofilament damage that is rapidly triggered by 10^-2M caffeine, 6x10^-6M ruthenium red, 10^-4M DNP or 5 g ml^-1 A23187. These agents also failed completely to protect against myofilament damage in saponin-skinned amphibian skeletal muscle when [Ca]_i was raised to 8x10^-6M. Thus, inhibition of PLA₂ does not protect the myofilament apparatus against calcium released intracellularly, and it is suggested that mepacrine and indomethacin can block entry of calcium in the calcium-paradox in the amphibian heart. Chlorpromazine (2x10^-4M) and mepacrine (10^-3M) at zero [Ca] caused severe myofilament damage in skinned muscle, possibly due to an effect on membranes. Since inhibitors of PLA₂ and of lipoxygenases prevent efflux of creatine kinase and sarcolemma damage in mammalian skeletal muscle, it is evident that experimentally-induced rises in [Ca]_i (by caffeine or A23187) can trigger two separate pathways: (i) PLA₂ and the arachidonic acid cascade which culminate in membrane damage, and (ii) a different, Ca-activated system that causes rapid damage of myofilaments.     

Characterisation and in vivo behaviour of a Salmonella typhimurium aroA strain expressing Escherichia coli K1 polysaccharide

Abstract Plasmid pKT274 encoding a determinant for the Escherichia coli K1 polysaccharide was introduced into the Salmonella typhimurium aro A vaccine strain SL3261 and cells harbouring the plasmid were shown to express K1 polysaccharide at their cell surface. SL3261 (pKT274) could be detected in the livers and spleens of BALB/c mice infected by the intravenous route and viable organisms persisted for several weeks. SL3261 (pKT274) was cleared from the livers more rapidly and from the spleens more slowly than SL3261. Unlike mice infected with SL3261 those infected with SL3261 (pKT274) did not exhibit gross splenomegaly during the first three weeks after infection. Mice vaccinated with viable SL3261 (pKT274) were protected against challenge with virulent S. typhimurium but failed to produce detectable levels of humoral anti-K1 polysaccharide antibodies.     

Drosophila basement membrane procollagen IV. I. Protein characterization and distribution

A collagen was isolated from Drosophila E85, Schneider line 2L and Kc cell cultures. The purified protein was characterized and antibodies were raised against it. Immunofluorescence microscopy locates this material to the regions of basement membranes of Drosophila embryos, larvae, and adults. The molecules are mostly, or entirely, homotrimers of one polypeptide chain linked by interchain disulfide bonds. The partial amino acid sequences of a cyanogen bromide cleavage product of this chain are identical with a part of the virtual translation product of the Drosophila pro alpha 1(IV) nucleotide sequence that is reported in the accompanying paper. This gene is at Drosophila chromosome location 25C and was identified by the high homology of one part of it with the noncollagenous carboxyl terminus (NC1) of vertebrate type IV basement membrane collagens (Blumberg, B., MacKrell, A. J., Olson, P. F., Kurkinen, M., Monson, J. M., Natzle, J. E., and Fessler, J. H. (1987) J. Biol. Chem. 262, 5947-5950). In the electron microscope each molecule appears as a thread with a knob at one end, which contains the carboxyl peptide domains. The variation of flexibility of the thread was mapped along its length. Pulse-chase labeling of cell cultures showed that these molecules associate into disulfide-linked dimers and higher oligomers that can be partly separated by velocity sedimentation and are resolved by sodium dodecyl sulfate-agarose gel electrophoresis. Dimers and higher oligomers formed by overlap of the amino ends of molecules were found. Mild pepsin digestion of Drosophila embryos and larvae solubilized the corresponding disulfide-linked collagen molecules, and Staphylococcus aureus V8 protease peptide maps showed the identity of the collagen derived from animals and from cell cultures. Individual, native molecules have a sedimentation coefficient s20,w = 4.1 S, the dichroic spectrum and amino acid composition of a collagen, and a Tm = 31 degrees C. Positive in situ hybridization with a specific probe for this collagen began 6-8 h after egg laying and showed message in the locations of embryos and larvae which reacted with the antibodies. This included some prominent individual cells in the hemolymph.     

Ultrastructural changes in the cardiomyopathy of dystrophic hamsters and mice

Changes in the ultrastructure of the cardiac muscle cells have been followed in dystrophic mice and hamsters (22-40 weeks of age) and in both species a severe cardiomyopathy accompanies the cellular damage of the skeletal muscle. The degradative changes of the myofilament apparatus of the heart cells and the specific changes in mitochondrial ultrastructure (including swelling, septation and apparent division) are characteristic of the cellular damage of both the dystrophic skeletal muscle and of normal cardiac muscle in which [Ca]i has been experimentally raised, confirming the suggestions that (i) the same gene is responsible for the myopathy of skeletal and cardiac muscle in animal dystrophy and (ii) that changes in [Ca]i are implicated in the degradative changes of muscle cells.     

Failure to detect changes in sarcolemma permeability in amphibian muscle following severe cellular damage

1. Isolated amphibian hearts and pectoris cutaneous muscles were exposed either to DNP or to caffeine, thereby producing severe myofilament damage. 2. No accompanying change in sarcolemma permeability was detected by monitoring either CK or LDH release or Procion yellow entry in the heart, or by Procion entry in amphibian skeletal muscle. 3. The findings are in contrast with mammalian cardiac and skeletal muscles, and confirm that the pathways leading to myofilament degradation and to the breakdown in sarcolemma organization are separate.     

Effect of aldosterone on 86Rb fluxes in cultured kidney cells (A6)

This study was designed to evaluate the relative contributions of hormone induced changes in active and passive K+ transport in an epithelial cell line in continuous culture derived from toad kidney (A6) using 86Rb as a tracer for measuring unidirectional K+ fluxes. The effects of 24 h exposure to aldosterone (A) and aldosterone plus insulin (A+I) on unidirectional K+ fluxes were evaluated under short-circuited conditions and under open circuit conditions. In epithelia exposed to A, a small but significant amount of active K+ secretion was found, although it was not significantly greater than in control epithelia. The bidirectional fluxes in both A and A+I treated epithelia, under short-circuited conditions, increased by a similar amount over control values indicating an increase in apparent permeability of passive transepithelial K+ transport. Under open circuit conditions, A stimulated net K+ transport by about 5-fold over controls. The increase in K+ secretion produced by A under open circuit conditions could be explained by the combined effects of an increase in transepithelial K+ permeability and an increase in the transepithelial electrical potential difference (PD). The presence of I produced no additional effects to that of A on K+ transport under the conditions used in this study. It is concluded that the substantial increase in K+ secretion induced in A6 cells by 24 h exposure to A is primarily passive in nature. It is possible that the changes in both PD and transepithelial K+ permeability, which can account for the observed increase in K+ secretion, are secondary to the stimulation of active Na+ transport.