A vertebrate slow skeletal muscle actin isoform

Salmonids utilize a unique, class II isoactin in slow skeletal muscle. This actin contains 12 replacements when compared with those from salmonid fast skeletal muscle, salmonid cardiac muscle and rabbit skeletal muscle. Substitutions are confined to subdomains 1 and 3, and most occur after residue 100. Depending on the pairing, the 'fast', 'cardiac' and rabbit actins share four, or fewer, substitutions. The two salmonid skeletal actins differ nonconservatively at six positions, residues 103, 155, 278, 281, 310 and 360, the latter involving a change in charge. The heterogeneity has altered the biochemical properties of the molecule. Slow skeletal muscle actin can be distinguished on the basis of mass, hydroxylamine cleavage and electrophoretic mobility at alkaline pH in the presence of 8 m urea. Further, compared with its counterpart in fast muscle, slow muscle actin displays lower activation of myosin in the presence of regulatory proteins, and weakened affinity for nucleotide. It is also less resistant to urea- and heat-induced denaturation. The midpoints of the change in far-UV ellipticity of G-actin versus temperature are approximately 45 degrees C ('slow' actin) and approximately 56 degrees C ('fast' actin). Similar melting temperatures are observed when thermal unfolding is monitored in the aromatic region, and is suggestive of differential stability within subdomain 1. The changes in nucleotide affinity and stability correlate with substitutions at the nucleotide binding cleft (residue 155), and in the C-terminal region, two parts of actin which are allosterically coupled. Actin is concluded to be a source of skeletal muscle plasticity.     

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.     

The 2'-O-methyltransferase responsible for modification of yeast tRNA at position 4

The methylation of the ribose 2'-OH of RNA occurs widely in nature and in all stable RNAs and occurs at five positions in yeast tRNA. 2'-O-methylation of tRNA at position 4 is interesting because it occurs in the acceptor stem (which is normally undermodified), it is the only 2'-O-methylation that occurs in the middle of a duplex region in tRNA, the modification is conserved in eukaryotes, and the features of the tRNA necessary for substrate recognition are poorly defined. We show here that Saccharomyces cerevisiae ORF YOL125w (TRM13) is necessary and sufficient for 2'-O-methylation at position 4 of yeast tRNA. Biochemical analysis of the S. cerevisiae proteome shows that Trm13 copurifies with 2'-O-methylation activity, using tRNAGlyGCC as a substrate, and extracts made from a trm13-Delta strain have undetectable levels of this activity. Trm13 is necessary for activity in vivo because tRNAs isolated from a trm13-Delta strain lack the corresponding 2'-O-methylated residue for each of the three known tRNAs with this modification. Trm13 is sufficient for 2'-O-methylation at position 4 in vitro since yeast Trm13 protein purified after expression in Escherichia coli has the same activity as that produced in yeast. Trm13 protein binds substrates tRNAHis and tRNAGlyGCC with KD values of 85+/-8 and 100+/-14 nM, respectively, and has a KM for tRNAHis of 10 nM, but binds nonsubstrate tRNAs very poorly (KD>1 microM). Trm13 is conserved in eukaryotes, but there is no sequence similarity between Trm13 and other known methyltransferases.     

In vitro studies of antifreeze glycoprotein (AFGP) and a C-linked AFGP analogue

Antifreeze glycoproteins (AFGPs) are a subclass of biological antifreezes found in deep sea Teleost fish. These compounds have the ability to depress the freezing point of the organism such that it can survive the subzero temperatures encountered in its environment. This physical property is very attractive for the cryopreservation of cells, tissues, and organs. Recently, our laboratory has designed and synthesized a functional carbon-linked (C-linked) AFGP analogue (1) that demonstrates tremendous promise as a novel cryoprotectant. Herein we describe the in vitro effects and interactions of C-linked AFGP analogue 1 and native AFGP 8. Our studies reveal that AFGP 8 is cytotoxic to human embryonic liver and human embryonic kidney cells at concentrations higher than 2 and 0.63 mg/mL, respectively, whereas lower concentrations are not toxic. The mechanism of this cytotoxicity is consistent with apoptosis because caspase-3/7 levels are significantly elevated in cell cultures treated with AFGP 8. In contrast, C-linked AFGP analogue 1 displayed no in vitro cytotoxicity even at high concentrations, and notably, caspase-3/7 activities were suppressed well below background levels in cell lines treated with 1. Although the results from these studies limit the human applications of native AFGP, they illustrate the benefits of developing functional C-linked AFGP analogues for various medical, commercial and industrial applications.     

A liquid chromatographic-tandem mass spectrometric method for the determination of two selective thymidylate synthase inhibitors, BGC945 and BGC638, in mouse plasma

A LC-tandem mass spectrometry method to quantify the quinazoline-based thymidylate synthase inhibitors BGC945 and BGC638 in mouse plasma was developed. BGC945 and BGC638 were extracted from mouse plasma using protein precipitation with acetonitrile. Chromatography was performed on a Fluophase RP 5 microm, 100 mmx2.0mm i.d. column using a gradient of ammonium acetate and acetonitrile as a mobile phase with a flow rate of 0.2 mLmin(-1). The injection volume for each sample was 20 microL with a total run time of 7.5 min. This method was validated in the range 25-4000 nM (r2=0.99). The analytical assay performance showed that the method was accurate (mean intra- and inter-day assay R.E. were below 12% and 11%, respectively), reproducible (mean intra- and inter-day R.S.D. were less than 13% and 5% for all quality control levels, respectively) and sensitive (lower limit of quantification was 25 nM) in the range studied. This validated method has been used to define the first pharmacokinetic report of BGC945 and BGC638 in mice.     

Analysis of cis-acting DNA elements mediating induction and repression of the spinach nitrite reductase gene

The expression of nitrite reductase (NiR; EC 1.7.7.1), the second enzyme in the nitrate assimilatory pathway, is regulated by nitrate as well as by end-products of nitrate assimilation, namely, glutamine (Gln) and asparagine (Asn). Nitrate induces expression of the NiR gene. Previously, using deletion analysis of the spinach (Spinacia oleracea L.) NiR gene promoter in transgenic tobacco (Nicotiana tabacum L.) and in-vivo dimethyl sulfate footprinting, we had identified the region between −230 bp and −180 bp as being critical for nitrate inducibility of this gene. In the present study, we show that the region from +1 to +67, which forms part of its untranslated leader, is important for minimal induction in the presence of nitrate. Electrophoretic mobility shift assays reveal concentration-dependent and competitive binding of a factor in tobacco nuclear extracts to this region. In the presence of Gln or Asn, the expression of spinach NiR is repressed. This repression is observed with the full-length NiR promoter (−3100 bp) as well as with the shortest promoter (−230 bp) that gives nitrate induction, which includes the +67 bp leader sequence. The repressed expression of the gene is not the result of reduced nitrate accumulation in the presence of the nitrogen metabolites. Received: 2 December 1997 / Accepted: 20 January 1998     

EVOLUTIONARY AND HISTORICAL ANALYSIS OF PROTEIN VARIATION IN THE BLOTCHED FORMS OF SALAMANDERS OF THE ENSATINA COMPLEX (AMPHIBIA: PLETHODONTIDAE)

Geographic variation in 23 to 29 protein-encoding genetic loci was examined in 48 populations of the Ensatina complex, a “ring species” distributed around the Central Valley of California. The samples span two critical links in the chain of morphologically distinct units: the transition from the unblotched to blotched color pattern types in the vicinity of Lassen Peak, northeastern California, and a geographic gap in the range of the complex in the San Gabriel Mountains, southern California. A general pattern of isolation by distance with a regular buildup of genetic distance correlated with increases in geographic distance characterizes the populations studied, with the exception of a little-differentiated group of populations in the northern Sierra Nevada; this region is postulated to be a zone of genetic reticulation characterized by relatively high gene flow. An adaptively significant color pattern is thought to have spread into the northern Sierra Nevada from the south, but protein variants have been introduced both from the north and the south. Genetic distances across the San Gabriel Mountain gap match expectations from the pattern of buildup of genetic distance as a function of geographic distance elsewhere in the complex. A phylogenetic analysis of the protein data supports the reticulation hypothesis; whereas the southernmost populations currently do constitute a monophyletic assemblage, an “extinction experiment” demonstrates that the distinction could be the result of the recent extinction of populations in a present gap in our sampling. The Ensatina complex appears to be a dynamic entity representing several stages in the evolution of species. It is a ring species, and whereas various taxonomic arrangements are possible, no taxonomic changes are proposed.     

Novel polymorphic microsatellite loci for distinguishing rock bass (<Emphasis Type="Italic">Ambloplites rupestris</Emphasis>), Roanoke bass (<Emphasis Type="Italic">Ambloplites cavifrons</Emphasis>), and their hybrids

The rock bass (Ambloplites rupestris) is a popular sport-fish native to the Mississippi and Great Lakes basins of North America. The species has been widely introduced outside its native range, including into Atlantic-slope streams of Virginia where it may hybridize with an imperiled, similar-looking congener, the Roanoke bass (Ambloplites cavifrons). In this study, we identified and evaluated novel molecular markers to facilitate identification of these species and study the extent of hybridization. Using molecular libraries developed from A. rupestris, we identified a suite of candidate nuclear microsatellite loci, synthesized primer sets, and tested these markers for amplification and polymorphism in populations of both species. We then calculated standard diversity statistics within and differentiation statistics between species, the latter providing an indication of marker power for distinguishing the species and their hybrids. Additionally, we evaluated our efficiency for identifying hybrids by classifying simulated genotypes of known ancestry. Eleven loci were polymorphic (2–22 alleles per locus) and reliably amplified in both species. Multilocus genetic differentiation between A. cavifrons and A. rupestris was quite high (F _ST  = 0.66; D _LR  = 19.3), indicating the high statistical power of this marker set for species and hybrid identification. Analyses of simulated data suggested these markers reliably distinguish between hybrids and non-hybrids, as well as between F1 hybrids and backcrossed individuals. This panel of 11 loci should prove useful for understanding patterns of hybridization between A. rupestris and A. cavifrons. As the first microsatellite markers developed for Ambloplites, these markers also should prove broadly useful for population genetic studies of this genus.