The ras oncogene and tumour metastasis: observations on murine cells transfected with activated human c-Ha-ras

Transfection of cells with cloned genes or total genomic DNA offers a means for studying aspects of neoplastic behaviour. We have used this method to examine whether incorporation of the cloned 6.6-kilobase (kb) fragment of DNA containing the mutant c-Ha-ras human oncogene can confer metastatic capability on murine NIH 3T3 cells. Cells co-transfected with the mutated ras gene and the neomycin resistance marker pSV2neo were selected by culture in neomycin. On subcutaneous inoculation into MF 1 nude mice, these cells proved to be tumourigenic with short latent periods (approximately 14 days)--nude mice were used to circumvent immunological rejection of the mouse cells expressing the product of the human oncogene. Transfectants were capable of lung colonisation after intravenous injection, but there was no evidence of spontaneous metastasis at autopsy, or on histological examination of the lungs and other organs, 90 days after inoculation. Incorporation of the transfected oncogene was confirmed by Southern blotting and its expression by dot-blot hybridisation and immunoprecipitation. The results in this experimental system indicate that transfection of a mutated human ras oncogene into non-neoplastic 3T3 cells can confer part of the metastatic phenotype, namely lung colonisation, but is not by itself sufficient to induce spontaneous metastatic behaviour.     

Reclustering of scattered Golgi elements occurs along microtubules

Depolymerization of the interphase microtubules by nocodazole results in the scattering and apparent fragmentation of the Golgi apparatus in Vero fibroblast cells. Upon removal of the drug, the interphase microtubules repolymerize, and the scattered Golgi elements move back to the region around the microtubule-organizing center (MTOC) within 40 to 60 min. Using a fluorescent lipid analogue (C6-NBD-ceramide) as a vital stain for the scattered Golgi elements, their relocation was visualized by video-enhanced fluorescence microscopy in Vero cells maintained at 20 degrees C. The NBD-labeled structures were identified as Golgi elements by their colocalization with galactosyltransferase in the fixed cells. During reclustering, NBD-labeled Golgi elements were observed to move by discontinuous saltations towards the MTOC with velocities of 0.1 to 0.4 micron/s. Paths along which Golgi elements moved were super-imposable on microtubules visualized by indirect immunofluorescence. Neither the collapse of intermediate filaments caused by microinjection of antibodies to vimentin nor the disruption of microfilaments by cytochalasin D had an effect on the reclustering of Golgi elements or the positioning of the Golgi apparatus. These data show that scattered Golgi elements move along microtubules back to the region around the MTOC, while neither intact intermediate filaments nor microfilaments are involved.     

Agarose gel electrophoresis in isozyme separation and visualization

Isozyme analysis of rodent-human somatic cell hybrids has been used frequently to detect specific human chromosomes. The majority of these isozyme systems employs starch gels, the use of which can be laborious when screening large numbers of cell lines. We describe the development of two procedures to detect the long arms of human chromosomes 1 and 2 in Chinese hamster-human cell hybrids by a rapid and reproducible method using 1-mm-thick agarose gels. Detection of human chromosome 1q was accomplished by screening for human fumarate hydratase activity, whose gene has been mapped to 1q42.1. Detection of chromosome 2q was performed by screening for the isozyme isocitrate dehydrogenase 1, which has been localized to 2q32-qter. These systems provide a basis for the further development of procedures for detecting chromosome-specific isozyme markers in agarose gels.     

Testosterone, and winning and losing in human competition

Testosterone and cortisol were measured in six university tennis players across six matches during their varsity season. Testosterone rose just before most matches, and players with the highest prematch testosterone had the most positive improvement in mood before their matches. After matches, mean testosterone rose for winners relative to losers, especially for winners with very positive moods after their victories and who evaluated their own performance highly. Winners with rising testosterone had higher testosterone before their next match, in contrast to losers with falling testosterone, who had lower testosterone before their next match. Cortisol was not related to winning or losing, but it was related to seed (top players having low cortisol), and cortisol generally declined as the season progressed. These results are consistent with a biosocial theory of status.     

Raman spectroscopic studies of hen egg-white lysozyme at high temperatures and pressures

In situ high-temperature, high-pressure Raman experiments on 3 mM (pH 5) aqueous solutions of hen egg-white (HEW) lysozyme show a decrease in the relative height of the 505 cm^–1 band associated with S-S stretching vibrations at 72°C (1 bar). The peak height changes are accompanied by significant band broadening, and the integrated band intensity does not change within experimental error. The effect of increased pressure at 72°C was to hinder broadening of the 505 cm^–1 band. HEW lysozyme (2.4 mM,pH 5) was also heated at 76°C, 80°C, and 95°C for different periods of time, and aliquots were quenched to room temperature for Raman and enzymatic activity measurements. After 9 hr at 76°C, the protein exhibits enzyme activity less than 50% of the initial value, and approximately 50% reduction in activity is achieved after 3 hr at 80°C or 1 hr at 95°C. The Raman results suggest that different irreversibly denatured conformations are attained during prolonged exposures at these different temperatures. It is apparent from these studies that the S-S stretch intensity is decreased irreversibly.     

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.