Diversity of Chlamydia trachomatis major outer membrane protein genes.

Genomic DNA libraries were constructed for Chlamydia trachomatis serovars B and C by using BamHI fragments, and recombinants that contained the major outer membrane protein (omp1) gene for each serovar were identified and sequenced. Comparisons between these gene sequences and the gene from serovar L2 demonstrated fewer base pair differences between serovars L2 and B than between L2 and C; this finding is consistent with the serologic and antigenic relationships among these serovars. The translated amino acid sequence for the major outer membrane proteins (MOMPs) contained the same number of amino acids for serovars L2 and B, whereas the serovar C MOMP contained three additional amino acids. The antigenic diversity of the chlamydial MOMP was reflected in four sequence-variable domains, and two of these domains were candidates for the putative type-specific antigenic determinant. The molecular basis of omp1 gene diversity among C. trachomatis serovars was observed to be clustered nucleotide substitutions for closely related serovars and insertions or deletions for distantly related serovars.     

ANOMALOUS CONTRACTION OF INVERTEBRATE STRIATED MUSCLE

The phenomenon of A band shortening or contraction has been investigated in glycerinated myofibrils of Pecten irradians, Homarus americanus, Cambarus virilis, and Limulus polyphemus through the techniques of ultraviolet microbeam inactivation and polarization microscopy. With the former method, it has been shown that these muscles, even though exhibiting the shortening effect, contract in a manner consistent with only the sliding filament model. Intrinsic birefringence studies have indicated no significant changes in mass distribution or orientation within the shortened A bands. Except in the case of Limulus muscle, the shortening effect was seen only in contraction under tension. The magnitude of this anomalous phenomenon was dependent upon glycerination time and has been duplicated in rabbit psoas muscle through brief trypsin treatment. A band shortening could not be observed in glutaraldehyde-fixed muscle or in myofibrils glycerinated for only short periods. It has been concluded that the phenomenon of A band contraction is an artifact induced by the glycerination procedure, possibly through weakening of the sarcomere structure. However, the fact that the A band shortens under tension rather than lengthens poses an interesting paradox.     

Plasminogen activation by t-PA on the surface of human melanoma cells in the presence of alpha 2-macroglobulin secretion.

Several human melanoma cell lines produced tissue-type plasminogen activator (t-PA), as detected by zymography and immunocapture assay of culture media and cell lysates. Urokinase (u-PA) was found at only less than or equal to 1% the level of t-PA. Acid eluates of the cell surface indicated that the melanoma cells had t-PA bound on their surface, but no u-PA, and also had a very low capacity to bind exogenous u-PA. After incubation of the melanoma cells with 10% plasminogen-depleted fetal calf serum and human plasminogen, bound plasmin activity could be eluted from the cell surface with tranexamic acid, an analogue of lysine. This indicated that plasminogen was activated on the cell surface. The cell-surface plasmin formation was inhibited by an anti-catalytic monoclonal antibody to human t-PA, and not by an anti-catalytic antibody to u-PA. The melanoma cells also synthesized and secreted alpha 2-macroglobulin (alpha 2M), as shown by alpha 2M-specific mRNA in Northern blotting and detection of alpha 2M protein in conditioned cell culture media. The media were found to inhibit u-PA but not t-PA. This inhibition was related to their alpha 2M content, and immunoabsorption of alpha 2M removed the inhibitory activity. These studies suggest that t-PA can bind to the surface of melanoma cells and generate surface-bound plasmin. Because t-PA and cell-bound plasmin are unaffected by alpha 2M, t-PA may, in the case of melanoma cells, serve an analogous function to u-PA in supporting tumor cell invasion.     

Genomics facilitates evaluation and monitoring of McCloud River Redband Trout (Oncorhynchus mykiss stonei)

The McCloud River Redband Trout (MRRT; Oncorhynchus mykiss stonei) is a unique subspecies of rainbow trout that inhabits the isolated Upper McCloud River of Northern California. A major threat to MRRT is introgressive hybridization with non-native rainbow trout from historical stocking and contemporary unauthorized introductions. To help address this concern, we collected RAD-sequencing data on 308 total individuals from MRRT and other California O. mykiss populations and examined population structure using Principal Component and admixture analyses. Our results are consistent with previous studies; we found that populations of MRRT in Sheepheaven, Swamp, Edson, and Moosehead creeks are nonintrogressed. Additionally, we saw no evidence of introgression in Dry Creek, and suggest further investigation to determine if it can be considered a core MRRT conservation population. Sheepheaven Creek was previously thought to be the sole historical lineage of MRRT, but our analysis identified three: Sheepheaven, Edson, and Dry creeks, all of which should be preserved. Finally, we discovered diagnostic and polymorphic SNP markers for monitoring introgression and genetic diversity in MRRT. Collectively, our results provide a valuable resource for the conservation and management of MRRT.

     

Phylogenetic patterns of trait and trait plasticity evolution: Insights from amphibian embryos

Environmental variation favors the evolution of phenotypic plasticity. For many species, we understand the costs and benefits of different phenotypes, but we lack a broad understanding of how plastic traits evolve across large clades. Using identical experiments conducted across North America, we examined prey responses to predator cues. We quantified five life‐history traits and the magnitude of their plasticity for 23 amphibian species/populations (spanning three families and five genera) when exposed to no cues, crushed‐egg cues, and predatory crayfish cues. Embryonic responses varied considerably among species and phylogenetic signal was common among the traits, whereas phylogenetic signal was rare for trait plasticities. Among trait‐evolution models, the Ornstein–Uhlenbeck (OU) model provided the best fit or was essentially tied with Brownian motion. Using the best fitting model, evolutionary rates for plasticities were higher than traits for three life‐history traits and lower for two. These data suggest that the evolution of life‐history traits in amphibian embryos is more constrained by a species’ position in the phylogeny than is the evolution of life history plasticities. The fact that an OU model of trait evolution was often a good fit to patterns of trait variation may indicate adaptive optima for traits and their plasticities.     

Alpha 2-macroglobulin restricts plasminogen activation to the surface of RC2A leukemia cells.

Human RC2A myelomonocytic leukemia cells are able to activate the prourokinase (pro-u-PA) they secrete so that active u-PA is present both in serum-free conditioned medium from these cells, as well as on the cell surface. When the cells are grown in serum-containing medium, no u-PA activity can be found in the medium but active u-PA is found bound to the cell surface where it can generate bound plasmin. This distribution of u-PA activity was shown to be, first, the net result of slow inactivation of free active u-PA by serum inhibitor(s) and simultaneous rapid uptake of u-PA onto the cell surface. Binding to cells was at least six times faster than inactivation by 10% serum. The principal serum inhibitor of u-PA was identified as alpha 2-macroglobulin (alpha 2M), and prior inactivation of u-PA by purified human alpha 2M was also shown to prevent uptake of u-PA activity onto cells. Second, although endogenous u-PA could form covalent complexes with purified alpha 2M in the culture medium of RC2A cells, covalent alpha 2M complexes were not formed by u-PA on the cell surface; the u-PA taken up in this compartment was protected against alpha 2M inhibition. u-PA anchored to plastic surfaces via monoclonal antibodies to the amino-terminal region of u-PA was also protected against alpha 2M, suggesting that the protection of cell surface u-PA results from a steric effect. These results provide evidence as to how the active u-PA produced by leukemia cells can contribute to proteolytic activity on their cell surface in the presence of serum inhibitors.     

Occurrence of 4-hydroxyalkenals in rat tissues determined as pentafluorobenzyl oxime derivatives by gas chromatography-mass spectrometry

Malondialdehyde measurements have been the major tool for studying relationships between lipid peroxidation and tissue pathology. Recently, we presented a novel gas chromatography-mass spectrometry method for direct detection of phospholipid peroxides with picogram sensitivity based on transesterification of phospholipids or triglycerides to form pentafluorobenzyl esters. Under some circumstances the reactive primary oxidation products break down. Therefore, we developed a convenient, high sensitivity method to detect more stable secondary lipid oxidation products, the 4-hydroxyalkenals. The method accomplishes a facile extraction of 4-hydroxynonenal from tissues by forming pentafluorobenzyl oxime derivatives to displace aldehydes from Schiff base linkages. 4-hydroxynonenal was found in heart, liver, adrenal, and testis from rats and was detected to the 10-100 pg level by the current method.     

Therapeutic antibody fragments with prolonged in vivo half-lives.

Antibody fragments can be isolated rapidly using techniques such as phage display and can be expressed to high levels in microbial systems. However, to date such antibody fragments have been of limited use for many therapeutic applications because they are rapidly cleared from the body. We present a strategy for the site-specific chemical modification of antibody fragments with polyethylene glycol, which results in the production of antibody fragments with long in vivo half-lives and full retention of antigen-binding properties. This technology should allow more rapid and economical production of therapeutic antibodies for chronic disease therapy.