Patterns of differentiation and hybridization in North American wolflike canids, revealed by analysis of microsatellite loci

Genetic divergence and gene flow among closely related populations are difficult to measure because mutation rates of most nuclear loci are so low that new mutations have not had sufficient time to appear and become fixed. Microsatellite loci are repeat arrays of simple sequences that have high mutation rates and are abundant in the eukaryotic genome. Large population samples can be screened for variation by using the polymerase chain reaction and polyacrylamide gel electrophoresis to separate alleles. We analyzed 10 microsatellite loci to quantify genetic differentiation and hybridization in three species of North American wolflike canids. We expected to find a pattern of genetic differentiation by distance to exist among wolflike canid populations, because of the finite dispersal distances of individuals. Moreover, we predicted that, because wolflike canids are highly mobile, hybrid zones may be more extensive and show substantial changes in allele frequency, relative to nonhybridizing populations. We demonstrate that wolves and coyotes do not show a pattern of genetic differentiation by distance. Genetic subdivision in coyotes, as measured by theta and Gst, is not significantly different from zero, reflecting persistent gene flow among newly established populations. However, gray wolves show significant subdivision that may be either due to drift in past Ice Age refugia populations or a result of other causes. Finally, in areas where gray wolves and coyotes hybridize, allele frequencies of gray wolves are affected, but those of coyotes are not. Past hybridization between the two species in the south-central United States may account for the origin of the red wolf.      

Fold-recognition and comparative modeling of human α2,3-sialyltransferases reveal their sequence and structural similarities to CstII from Campylobacter jejuni

Background  

The 3-D structure of none of the eukaryotic sialyltransferases (SiaTs) has been determined so far. Sequence alignment algorithms such as BLAST and PSI-BLAST could not detect a homolog of these enzymes from the protein databank. SiaTs, thus, belong to the hard/medium target category in the CASP experiments. The objective of the current work is to model the 3-D structures of human SiaTs which transfer the sialic acid in α2,3-linkage viz., ST3Gal I, II, III, IV, V, and VI, using fold-recognition and comparative modeling methods. The pair-wise sequence similarity among these six enzymes ranges from 41 to 63%.     

Influence of the ventral surface of the medulla on tracheal responses to CO2

These studies investigated the role of the intermediate area of the ventral surface of the medulla (VMS) in the tracheal constriction produced by hypercapnia. Experiments were performed in chloralose-anesthetized, paralyzed, and artificially ventilated cats. Airway responses were assessed from pressure changes in a bypassed segment of the rostral cervical trachea. Hyperoxic hypercapnia increased tracheal pressure and phrenic nerve activity. Intravenous atropine pretreatment or vagotomy abolished the changes in tracheal pressure without affecting phrenic nerve discharge. Rapid cooling of the intermediate area reversed the tracheal constriction produced by hypercapnia. Graded cooling produced a progressive reduction in the changes in maximal tracheal pressure and phrenic nerve discharge responses caused by hypercapnia. Cooling the intermediate area to 20 degrees C significantly elevated the CO2 thresholds of both responses. These findings demonstrate that structures near the intermediate area of the VMS play a role in the neural cholinergic responses of the tracheal segment to CO2. It is possible that neurons or fibers in intermediate area influence the motor nuclei innervating the trachea. Alternatively, airway tone may be linked to respiratory motor activity so that medullary interventions that influence respiratory motor activity also alter bronchomotor tone.     

Effect of N-methyl-D-aspartate applied to the ventral surface of the medulla on the trachea

Structures located near the ventral surface of the medulla (VMS) affect both cardiovascular tone and respiratory activity. In addition cooling the intermediate area of the VMS blocks the increases in parasympathetic activity and tracheal tone resulting from ventilation with hypercapnic or hypoxic gas mixtures, or due to stimulation of mechanoreceptors within the lung. Since cooling the surface of the VMS may affect fibers of passage as well as cell bodies, we performed studies in which pledgets containing N-methyl-D-aspartic acid (NMDA), a synthetic excitatory amino acid, were applied to intermediate area of the VMS. The studies were performed in chloralose-anesthetized, artificially ventilated cats. Application of pledgets containing NMDA (10(-7) mol at 10(-3) M) caused increases in tracheal pressure and the onset of phasic phrenic activity, but application of 10(-8) mol at 10(-4) M of NMDA could produce tracheal constriction without the appearance of phasic phrenic activity. Applying to the entire VMS either 2-amino-5-phosphonovalerate (2-APV, 10(-6) M), a specific antagonist to NMDA, or lidocaine (2%), a local anesthetic, 60 s before the application of pledgets containing NMDA, prevented the increase in tracheal tone and phasic phrenic activity. Intravenous administration of atropine methyl nitrate 0.5 mg/kg, a cholinergic antagonist, blocked tracheal responses to local application of pledgets containing NMDA but did not affect the increase in phasic phrenic nerve activity. These findings suggest that when stimulated, neurons near the surface of the VMS in the vicinity of the intermediate area increase the activity of parasympathetic fibers to the airway.     

Antifilarial and Antibiotic Activities of Methanolic Extracts of Melaleuca cajuputi Flowers

We evaluated the activity of methanolic extracts of Melaleuca cajuputi flowers against the filarial worm Brugia pahangi and its bacterial endosymbiont Wolbachia. Anti-Wolbachia activity was measured in worms and in Aedes albopictus Aa23 cells by PCR, electron microscopy, and other biological assays. In particular, microfilarial release, worm motility, and viability were determined. M. cajuputi flower extracts were found to significantly reduce Wolbachia endosymbionts in Aa23 cells, Wolbachia surface protein, and microfilarial release, as well as the viability and motility of adult worms. Anti-Wolbachia activity was further confirmed by observation of degraded and phagocytized Wolbachia in worms treated with the flower extracts. The data provided in vitro and in vivo evidence that M. cajuputi flower extracts inhibit Wolbachia, an activity that may be exploited as an alternative strategy to treat human lymphatic filariasis.     

Biochemical purification and crystallographic characterization of the fiber-forming protein pilin from Neisseria gonorrhoeae

Pilus fibers are long protein filaments on many pathogenic bacteria that participate in attachment to host cells. Although the self-assembling protein pilin is the major structural component of the Neisseria gonorrhoeae pilus fiber, several other proteins co-purified with pilin through the repeated solubilization-reassociation steps of the biochemical purification. Pilin solubilized in the nondenaturing detergent n-octyl-beta-D-glucopyranoside remained an aggregate of about 100 kDa at pH 9.5, but was reduced to a 40-kDa dimer at pH 10.5, suggesting that assembly involves electrostatic interactions of lysine, tyrosine, or other side chains with high pKa values. Pilin dimers and aggregates of higher molecular mass were partially stable even in the presence of sodium dodecyl sulfate and beta-mercaptoethanol. Removal of pilus-associated proteins and stabilization of pilin multimers permitted the reproducible crystallization of pilin. Three-dimensional needle- and plate-shaped crystals of purified N. gonorrhoeae pilin (strain MS11 variant C30) grew from 36 to 40% polyethylene glycol 400, pH 8.0-9.0, in space group C222, with cell dimensions a = 126.4, b = 121.2, c = 26.7 A and Vm = 2.84 A3/dalton for one molecule per asymmetric unit. The best crystals diffracted to 2.4 A resolution using synchrotron radiation, were stable to x-ray damage, and appear suitable for determination of the atomic structure. This approach of stabilizing and crystallizing an intermediate assembly state may be useful for other fiber-forming proteins, which have previously not been successfully crystallized in forms that diffract to atomic resolution.     

Effect of freezing and thawing on a moderately halophilic bacterium as a function of Na+, K+, and Mg2+ concentration

A moderately halophilic bacterium was studied with respect to its ability to survive freezing and thawing as a function of sodium, potassium, and magnesium concentrations and rates of cooling and warming.Survival varied more strongly as a function of the warming rate than as a function of the cooling rate, and in general survivial increased with increasing molarity. At low overall molarity, sodium and potassium were largely interchangeable, while at high molarity, high potassium to sodium ratios improved survival. Survival at slow wanning rates was very poor, but protection by potassium was more pronounced.Magnesium proved protective, particularly where survival was otherwise poor. The protection provided by magnesium resulted in a decrease in the dependency on the cooling rate and, to a lesser extent, on the warming rate.