Evolution of the 28S ribosomal RNA gene in anurans: regions of variability and their phylogenetic implications

Fifteen restriction sites were mapped to the 28S ribosomal RNA gene of individuals representing 54 species of frogs, two species of salamanders, a caecilian, and a lungfish. Eight of these sites were present in all species examined, and two were found in all but one species. Alignment of these conserved restriction sites revealed, among anuran 28S rRNA genes, five regions of major length variation that correspond to four of 12 previously identified divergent domains of this gene. One of the divergent domains (DD8) consists of two regions of length variation separated by a short segment that is conserved at least throughout tetrapods. Most of the insertions, deletions, and restriction-site variations identified in the 28S gene will require sequence-level analysis for a detailed reconstruction of their history. However, an insertion in DD9 that is coextensive with frogs in the suborder Neobatrachia, a BstEII site that is limited to representatives of two leptodactylid subfamilies, and a deletion in DD10 that is found only in three ranoid genera are probably synapomorphies.      

An enzyme with properties similar to those of beta-N-acetylhexosaminidase S is expressed in the promyelocytic cell line HL-60.

Extracts of the human promyelocytic cell line HL-60 contain a form of beta-N-acetylhexosaminidase that is not retained on columns of benzeneboronate-agarose ('phenylboronate-agarose') and has a pI value lower than that of beta-N-acetylhexosaminidase A. It is clearly distinct from beta-N-acetylhexosaminidase A in its behaviour on DEAE-cellulose columns, and it requires a higher concentration of salt for its elution. This 'extra' form has a higher ratio of activity towards 4-methylumbelliferyl beta-N-acetylglucosaminide 6-sulphate and 4-methylumbelliferyl beta-N-acetylglucosaminide than has beta-N-acetylhexosaminidase A and is less stable when heated at 50 degrees C. It has a pH optimum of 4.5 and is therefore not beta-N-acetylglucosaminidase C. Anti-(human beta-N-acetylhexosaminidase alpha-subunit) serum precipitated both beta-N-acetylhexosaminidase A and the 'extra' form, whereas an anti-(beta-subunit) serum precipitated beta-N-acetylhexosaminidase A but not the 'extra' form. Western blotting and immunodetection of polypeptides derived from the 'extra' form revealed a band corresponding in size to mature alpha-subunits. On the basis of this and of its behaviour on isoelectric focusing, chromatofocusing and its kinetic properties, we conclude that the 'extra' form is composed of alpha-subunits and resembles beta-N-acetylhexosaminidase S, the residual form in Sandhoff's disease.     

Detecting base pair substitutions in DNA fragments by temperature-gradient gel electrophoresis.

A vertical gel electrophoresis apparatus is described which can distinguish DNA fragments differing by single base pair substitutions. The system employs a homogenous polyacrylamide gel containing urea-formamide and a temperature gradient which runs either perpendicular or parallel to the direction of electrophoresis. The temperature-gradient system simplifies several features of the denaturant-gradient system (1) and is relatively inexpensive to construct. Eight homologous 373 bp DNAs differing by one, two, or nine base pair substitutions were examined. DNA electrophoretic mobility changed abruptly with the temperature induced unwinding of DNA domains. GC to AT substitutions at different locations within the first melting domain, as well as an AT to TA transversion were separated with temperature gradients parallel to the electrophoretic direction. The relative stabilities of the DNAs observed in the gels were compared to predictions of DNA melting theory. General agreement was observed however complete correspondence was not obtained.     

Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.     

Modulation of rat granulocyte traffic by a surface active agent in vitro and bleomycin injury

Pluronic F68 (F68) is a nonionic surfactant which has been reported to inhibit the in vitro adherence and migration of polymorphonuclear leukocytes (PMN) obtained from some species. We demonstrated similar effects on PMN obtained from rats, with diminished adherence to nylon wool and diminished chemotaxis toward zymosan-activated serum. We then examined the in vivo effects of 12-hr F68 infusion on the injury induced by intratracheal bleomycin instillation (ITB) in rats. When sacrificed 24 hr following injury, rats demonstrated neutrophilia, neutrophil-prominent lung lavage cellularity, and increased lung weights. F68 decreased lavage leukocyte counts and lung weight gain in ITB-injured animals. Lung weights of ITB-injured animals correlated (r = 0.81, P less than 0.001) with logarithmic values of lavage PMN. F68 also enhanced neutrophilia and decreased spleen weight gain in injured animals. The acute effects of F68 on circulating leukocyte counts, osmolality, and total complement were also examined. The data demonstrate that F68 can affect PMN traffic both in vitro and in vivo. The data also confirm the prominence of PMN in lavage fluid early in ITB injury, and suggest that an influx of relatively few PMN is associated with lung weight gain in this model.     

Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

Study of quercetin and fisetin synergistic effect on breast cancer and potentially involved signaling pathways

Naturally-derived drugs have drawn much attention in recent decades. Efficiency, lower toxicity, and economic reasons are some of their advantages that justify this broad range of administration for different diseases, including cancer. If we can find a specific combination that boosts the effects of their single therapy, leading to synergism effect, increased efficiency, and decreased toxicity, they can act even better. Quercetin and fisetin, two well-known flavonoids, have been used to fight against various cancers. In this study, we investigated their possible synergism quercetin and fisetin on MCF7, MDA-MB-231, BT549, T47D, and 4T1 breast cancer cell lines. Then the optimum combined dose was used to study their impacts on wound healing abilities and clonogenic properties. The real-time qPCR was used to study the expression of their validated downstream effectors in predicted pathways. A significant synergism effect (p < .01, combination index: <1) was observed for all cell lines. Combination therapy was significantly more effective in colony formation (p < .0001) and wound healing assays (p < .001) compared to single therapies. The expression level of potential effectors was also showed a greater change. In vivo study confirmed the in vitro results and showed how significantly (p < .001) their synergism promotes their singular function in inhibiting cancer progression. The breast cancer mouse models receiving combined therapy lived longer with higher average body weight and smaller tumor sizes. These results exhibit that quercetin and fisetin inhibit cancer cell proliferation, migration and colony formation synergistically, and matrix metalloproteinase signaling and apoptotic pathways are relatively responsible for inhibitory activities.     

Evidence for "twisted plane" undulations in golden hamster sperm tails

Motile spermatozoa from the golden hamster have been arrested by rapid freezing and then fixed with glutaraldehyde at low temperature after substitution with ethylene glycol. As far as can be judged, the flagellar waveforms thus stabilized are similar to those seen in living sperm; in contrast, fixation in glutaraldehyde, without prior freezing, induces agonal changes in flagellar conformation. The characteristics waveform after freeze substitution contains three bends. Approx. half of these flagella are entirely planar. The rest are three dimensional, with the third bend displaced in a regular way from the plane containing the proximal two bends. From the geometry of these flagella, it is concluded that the plane of action of a given bending cycle undergoes a clockwise twist (from a forward viewpoint) as the cycle is succeeded by new bending cycles. This "twisted plane" undulation is quite different from helical movement. The twisting seems to occur abruptly, between cycles, as if each bending cycle has a preferred plane of action. The mechanism underlying the twisting is uncertain. However, on the basis of the angular displacements between the preferred planes, and the findings from electron microscopy, the following idea is presented as a working hypothesis: that, if the most proximal plane of bending is topographically determined by peripheral doublet 1, then successive distal planes of action are influenced predominantly by doublets 2, 3, etc., in clockwise sequence. The merits and weaknesses of this hypothesis are discussed.