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.      

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.     

Neuronal origin of a cerebral amyloid: neurofibrillary tangles of Alzheimer''s disease contain the same protein as the amyloid of plaque cores and blood vessels.

The protein component of Alzheimer's disease amyloid [neurofibrillary tangles (NFT), amyloid plaque core and congophilic angiopathy] is an aggregated polypeptide with a subunit mass of 4 kd (the A4 monomer). Based on the degree of N-terminal heterogeneity, the amyloid is first deposited in the neuron, and later in the extracellular space. Using antisera raised against synthetic peptides, we show that the N terminus of A4 (residues 1-11) contains an epitope for neurofibrillary tangles, and the inner region of the molecule (residues 11-23) contains an epitope for plaque cores and vascular amyloid. The non-protein component of the amyloid (aluminum silicate) may form the basis for the deposition or amplification (possible self-replication) of the aggregated amyloid protein. The amyloid of Alzheimer's disease is similar in subunit size, composition but not sequence to the scrapie-associated fibril and its constituent polypeptides. The sequence and composition of NFT are not homologous to those of any of the known components of normal neurofilaments.     

Purification and characterization of succinyl-CoA: tetrahydrodipicolinate N-succinyltransferase from Escherichia coli

Tetrahydrodipicolinate succinylase, an enzyme involved in the diaminopimelate-lysine pathway, was purified 1900-fold from crude extracts of Escherichia coli. The enzyme catalyzes the formation of CoA and N-succinyl-2-amino-6-keto-L-pimelate from succinyl-CoA and tetrahydrodipicolinate. The purified enzyme was shown to be homogeneous by polyacrylamide gel electrophoresis. The Stokes radius of the enzyme was determined from its elution volume on a Sephacryl S300 column and its sedimentation constant from sucrose density gradient centrifugation. These were 35 A and 4.7 (S20,w), respectively. The enzyme consists of two subunits each with a mass of 31,000 daltons, as determined using sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Tetrahydrodipicolinate succinylase was shown to be a sulfhydryl enzyme. It has a pH optimum of 8.2. The equilibrium lies predominantly in favor of product formation but the reverse reaction can be demonstrated in vitro.     

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.      

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.     

Occurrence of ornithine decarboxylase and polyamines in cartilage.

The activity of ornithine decarboxylase was investigated in cartilage from chick embryos, rabbits, rats and human foetuses. The enzyme activity in these cartilages was of the same order as the detected in other body tissues. Ornithine decarboxylase activity in chick-embryo cartilage and liver was the same when compared on the basis of total soluble tissue protein. The cartilage enzyme exhibited a pH optimum of 6.5 and a Km for ornithine of 0.16mM. Ornithine decarboxylase activity in chick-embryo pelvic leaflets was maintained at the value in vivo for up to 22h when the isolated tissue was incubated in a modified Waymouth's medium (MB 752/1) at 37 degrees C. After addition of cycloheximide to the incubation medium, ornithine decarboxylase activity declined, with a half-life of 40 min. The concentrations of the polyamines spermidine and spermine in chick-embryo pelvic cartilage and rabbit costal cartilage were of the same order as the concentrations detected in other tissues.     

The effects of IL-1, IL-2, and tumor necrosis factor on polymorphonuclear leukocyte Fc gamma receptor-mediated phagocytosis. IL-2 down-regulates the effect of tumor necrosis factor

It has been reported that the Fc gamma R-mediated phagocytic activity of polymorphonuclear leukocytes (PMN) from patients with acute bacterial infections is markedly enhanced when compared with healthy controls. Inasmuch as several potent cytokines are known to be involved in inflammatory and infectious processes, we studied the effects of three such cytokines (IL-1 beta, IL-2, and TNF-alpha) on normal PMN Fc gamma R-mediated phagocytosis. IL-1 beta and TNF alpha both caused a significant increase in the ingestion of EIgG by adherent PMN. In combination, IL-1 beta and TNF-alpha had an additive effect, even when each was used at its optimal concentration. In contrast to the enhancing effects mediated by IL-1 beta and TNF-alpha, IL-2 alone had no significant effect on PMN phagocytosis. Notably, however, IL-2 at a concentration of 10(4) U/ml partially inhibited TNF-alpha-mediated enhancement of phagocytosis by decreasing TNF binding to the PMN cell surface. This inhibitory effect of IL-2 on TNF was reversed by anti-IL-2 antibody and mAb directed against the low affinity IL-2R (anti-Tac), whereas mAb directed against the intermediate affinity receptor (mik-beta 1) had no such effect. These findings may have important physiologic implications, because patients receiving IL-2 therapy have been shown to have increased susceptibility to infection.