High resolution mechanical function in the intact porcine heart: mechanical effects of pacemaker location

The necessity to quantify the mechanical function with high spatial resolution stemmed from the advancement of myocardial salvaging techniques. Since these therapies are localized interventions, a whole field technique with high spatial resolution was needed to differentiate the normal, diseased, and treated myocardium. We developed a phase correlation algorithm for measuring myocardial displacement at high spatial resolution and to determine the regional mechanical function in the intact heart. Porcine hearts were exposed and high contrast microparticles were placed on the myocardium. A pressure transducer, inserted into the left ventricle, synchronized the pressure (LVP) with image acquisition using a charge-coupled device camera. The deformation of the myocardium was measured with a resolution of 0.58+/-0.04 mm. Within the region of interest (ROI), regional stroke work (RSW), defined as the integral of LVP with respect to regional area, was determined on average at 21 locations with a resolution of 27.1+/-2.7 mm2. To alter regional mechanical function, the heart was paced at three different locations around the ROI. Independent of the pacemaker location, RSW decreased in the ROI. In addition, a gradient of increasing RSW in the outward direction radiating from the pacemaker was observed in all pacing protocols. These data demonstrated the ability to determine regional whole field mechanical function with high spatial resolution, and the significant alterations induced by electrical pacing.     

The structure of xylem vessels in grapevine (Vitaceae) and a possible passive mechanism for the systemic spread of bacterial disease

Xylem-dwelling pathogens become systemic, suggesting that microorganisms move efficiently in the xylem. To better understand xylem pathways and how bacteria move within the xylem, vessel connectivity between stems and leaves of Vitis vinifera cv. Chardonnay and Muscadinia rotundifolia cv. Cowart was studied. Three methods were used: (1) the light-producing bacterium, Yersinia enterocolitica, (Ye) strain GY5232 was loaded into petioles and followed using X-ray film, (2) fluorescent beads were loaded and followed by microscopy, and (3) low-pressure air was pumped into leaves and extruded bubbles from cuts in submerged leaves were followed. Bacteria, beads, and air moved through long and branched xylem vessels from the petiole into the veins in leaves of both varieties. From the stem, bacteria and air traveled into primary and secondary veins of leaves one, two, and three nodes above the loading point of the bacteria or air. Particles and air could move unimpeded through single xylem vessels or multiple vessels (conduits) connected possibly through broken pit membranes from within the stem axis into leaf blades. Bacteria were also able to move long distances within minutes from stem to leaf passively without having to cross pit membranes. Such complex, open xylem conduits have not been well documented before; these findings will help elucidate mechanisms involved in the systemic spread of pathogens.     

Hindlimb adaptations in Ourayia and Chipetaia, relatively large-bodied omomyine primates from the Middle Eocene of Utah

North American omomyids represent a tremendous Eocene radiation of primates exhibiting a wide range of body sizes and dietary patterns. Despite this adaptive diversity, relatively little is known of the postcranial specializations of the group. Here we describe hindlimb and foot bones of Ourayia uintensis and Chipetaia lamporea that were recovered from the Uinta B member (early Uintan Land Mammal Age), Uinta Formation, Utah. These specimens provide insights into the evolution of postcranial adaptations across different body sizes and dietary guilds within the Eocene primate radiation. Body mass estimates based on talar measurements indicate that Ourayia uintensis and Chipetaia lamporea weighed about 1,500-2,000 g and 500-700 g, respectively. Skeletal elements recovered for Ourayia include the talus, navicular, entocuneiform, first metatarsal, and proximal tibia; bones of Chipetaia include the talus, navicular, entocuneiform, and proximal femur. Both genera had opposable grasping big toes, as indicated by the saddle-shaped joint between the entocuneiform and first metatarsal. Both taxa were arboreal leapers, as indicated by a consistent assemblage of characters in all represented bones, most notably the somewhat elongated naviculars, the high and distinct trochlear crests of the talus, the posteriorly oriented tibial plateau (Ourayia), and the cylindrical head of the femur (Chipetaia). The closest resemblances to Ourayia and Chipetaia are found among the Bridger omomyines, Omomys and Hemiacodon. The results of our comparisons suggest that the later, larger, more herbivorous omomyines from Utah retained a skeletal structure characteristic of earlier, smaller North American omomyids.     

Coordinated alteration of hepatic gene expression in fatty acid and triglyceride synthesis in LCAT-null mice is associated with altered PUFA metabolism

Complete lecithin:cholesterol acyltransferase (LCAT) deficiency is associated with fasting hypertriglyceridemia (HTG). We recently reported that, in ldlr(-/-)xlcat(-/-) mice, fasting HTG is associated with hepatic triglyceride overproduction in association with an upregulation of the hepatic srebp1 gene and altered expression of its target genes in lipogenesis and gluconeogenesis. We further investigated the role of hepatic polyunsaturated fatty acid (PUFA) metabolism in the modulation of the lipid phenotypes. In the ldlr(-/-)xlcat(-/-) mice, using the ldlr(-/-)xlcat(+/+) littermate as controls, the hepatic level of cholesterol esters (CE) were reduced by 61.0% whereas the 20:4-CE and 22:6-CE contents were each reduced by >80%. In contrast, the hepatic levels of 20:4- and 22:6-containing phospholipid (PL) species were either unchanged or mildly elevated. Similar alterations of the hepatic PUFA in CE and in PL were also observed in the lcat(-/-) mice compared with their wild-type controls. In ldlr(-/-)xlcat(-/-) mice, hepatic mRNA level was markedly reduced for Delta-6 desaturase (fads2) (70.2%) and acyl-CoA:cholesterol acyltransferase-2 (soat2) (57.0%). A similar pattern of gene expression change was also observed in the lcat(-/-) single-knockout mice. In contrast, the acyl-CoA:diacylglycerol acyltransferase-2 (dgat2) mRNA level was 1.7-fold upregulated in the double-knockout mice. In summary, we observed coordinated alterations in hepatic expression of the gene for fads2, soat2, and dgat2, resulting in a reduction in total hepatic PUFA pool and differentially in the PUFA-CE pool, in association with an increase in dgat2 gene expression for promoting triglyceride synthesis and secretion. Some of the phenotypes are not readily explained by known mechanisms and may represent novel regulatory pathways.     

Development of polymorphic expressed sequence tag-derived microsatellites for the extension of the genetic linkage map of the black tiger shrimp (Penaeus monodon)

In this study, microsatellite markers were developed for the genetic linkage mapping and breeding program of the black tiger shrimp Penaeus monodon. A total of 997 unique microsatellite-containing expressed sequence tags (ESTs) were identified from 10 100 EST sequences in the P. monodon EST database. AT-rich microsatellite types were predominant in the EST sequences. Homology searching by the blastn and blastx programs revealed that these 997 ESTs represented 8.6% known gene products, 27.8% hypothetical proteins and 63.6% unknown gene products. Characterization of 50 markers on a panel of 35-48 unrelated shrimp indicated an average number of alleles of 12.6 and an average polymorphic information content of 0.723. These EST microsatellite markers along with 208 other markers (185 amplified fragment length polymorphisms, one exon-primed intron-crossing, six single strand conformation polymorphisms, one single nucleotide polymorphism, 13 non-EST-associated microsatellites and two EST-associated microsatellites) were analysed across the international P. monodon mapping family. A total of 144 new markers were added to the P. monodon maps, including 36 of the microsatellite-containing ESTs. The current P. monodon male and female linkage maps have 47 and 36 linkage groups respectively with coverage across half the P. monodon genome.     

Retrospective evaluation of the incidence and severity of hemosiderosis in a large captive lemur population

Significant concern has been generated about the susceptibility of captive lemurs to iron storage disease, which has led some researchers to propose husbandry changes regarding dietary iron. In the current study we sought to determine the history, severity, and prevalence of iron storage disease within a large captive lemur population. Iron concentration and hemosiderin accumulation in a target organ, the liver, were assessed in necropsy specimens from 15 different species (n=153) of lemurs over a 12-yr period at the Duke University Primate Center. Banked liver tissue was used to quantify liver iron concentration (LIC) via neutron activation analysis (NAA). Prussian blue staining was used to accentuate the presence of liver iron for evaluation using an established scoring system. Of the 153 reports examined, 49 (32%) of the animals were considered positive for the presence of hemosiderin in the liver, lymph node, duodenum, and kidney, with 36 of the 49 (73%) showing deposition of iron in the liver. Total iron scores (TIS) ranged from 0.3+/-0.3 in Lemur catta to 33.3+/-1.7 in Cheirogaleus medius. The mean LIC ranged from 209+/-1.4 microg/g wet weight in L. catta to 2957+/-414 microg/g in C. medius. Management practices may have contributed to some of the results observed in this study. Although evidence of excess iron deposition in the liver was present across several species studied, the levels were not as pervasive as previously reported in other captive lemur populations. Hemochromatosis was not observed, and excess iron was not related to the cause of death in any of the animals studied. The current findings suggest that iron overload in lemurs may be more complex than was previously believed.     

Comparison of serum iron, total iron binding capacity, ferritin, and percent transferrin saturation in nine species of apparently healthy captive lemurs

Lemurs kept in captivity have been reported to be highly prone to accumulate excessive amounts of iron in tissues (hemosiderosis). Diagnosis of the condition is most commonly made during a postmortem examination because an antemortem diagnosis requires a liver biopsy, a procedure that may not be well tolerated by all animals. The lack of a noninvasive method to evaluate iron status in captive lemurs limits investigators' ability to effectively screen animals for the presence of hemosiderosis, and to detect the condition early when treatment protocols are most effective. This study was conducted in an effort to provide data regarding iron analyte values in healthy captive lemurs of multiple species. The relationship of various iron-related metabolites was evaluated in 177 clinically normal lemurs of nine different species. Serum iron (sI), total iron binding capacity (TIBC), and ferritin concentration were measured directly and the percent transferrin saturation (TS) was calculated. Significant differences in various iron metabolites were observed among several species, suggesting that normal reference values for iron metabolites in lemurs may need to be developed on a species by species basis.     

Autofeedback effects of progressively rising oxytocin concentrations on supraoptic oxytocin neuronal activity in slices from lactating rats

Suckling stimuli induce somatodendritic oxytocin (OT) release from supraoptic nucleus (SON) neurons, which raises intranuclear OT concentrations and contributes to the effectiveness of the milk-ejection reflex. To clarify how such changes in OT concentrations modulate the activity of OT neurons, we examined OT effects using whole cell patch-clamp recordings from SON neurons in slices from lactating rats. Progressive increases from extremely low OT concentrations (0.1-10 fM) to high concentrations (0.1-10 nM) induced excitation and subsequent spike frequency reduction (SFR) in OT neurons. Significant effects of OT on firing rates were observed starting at 1 fM, reached peak level from 1 fM to 1 pM before SFR occurred in most neurons. The buildup of OT concentrations progressively promoted depolarization of membrane potential, spike broadening, decreases in spike amplitude, and increases in the rise time of spike afterhyperpolarizations, which were unrelated to firing rate. However, intermittent application of OT (1 fM, 1 pM, and 1 nM, each for 5 min) evoked dose-dependent excitation but not the SFR. Application of 1 pM OT for 40 min simulated the effects of progressively increasing OT concentrations. Vasopressin neurons were also activated by OT but did not show SFR. Consistent with presynaptic loci of OT action, ionotropic glutamate receptor antagonists reduced OT effects on firing rate, whereas bicuculline did not change the excitatory effects. These results suggest that the specific autoregulatory effects of OT, and perhaps other neuropeptides as well, are time and concentration dependent.     

Expression and characterization of a functional canine variant of cytochrome b5 reductase

Cytochrome b5 reductase (cb5r), a member of the flavoprotein transhydrogenase family of oxidoreductase enzymes, catalyzes the transfer of reducing equivalents from the physiological electron donor, NADH, to two molecules of cytochrome b5. We have determined the correct nucleotide sequence for the putative full-length, membrane-associated enzyme from Canis familiaris, and have generated a heterologous expression system for production of a histidine-tagged variant of the soluble, catalytic diaphorase domain, comprising residues I33 to F300. Using a simple two-step chromatographic procedure, the recombinant diaphorase domain has been purified to homogeneity and demonstrated to be a simple flavoprotein with a molecular mass of 31,364 (m/z) that retained both NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities. The recombinant protein contained a full complement of FAD and exhibited absorption and CD spectra comparable to those of a recombinant form of the rat cytochrome b5 reductase diaphorase domain generated using an identical expression system, suggesting similar protein folding. Oxidation-reduction potentiometric titrations yielded a standard midpoint potential (Eo') for the FAD/FADH2 couple of -273+/-5 mV which was identical to the value obtained for the corresponding rat domain. Thermal denaturation studies revealed that the canine domain exhibited stability comparable to that of the rat protein, confirming similar protein conformations. Initial-rate kinetic studies revealed the canine diaphorase domain retained a marked preference for NADH versus NADPH as reducing substrate and exhibited kcat's of 767 and 600 s(-1) for NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities, respectively, with Km's of 7, 8, and 12 microM for NADH, K3Fe(CN)6, and cytochrome b5, respectively. Spectral-binding constants (Ks) determined for a variety of NAD+ analogs indicated the highest and lowest affinities were observed for APAD+ (Ks=71 microM) and PCA+ (Ks=>31 mM), respectively, and indicated the binding contributions of the various portions of the pyridine nucleotide. These results provide the first correct sequence for the full-length, membrane-associated form of C. familiaris cb5r and provide a direct comparison of the enzymes from two phylogenetic sources using identical expression systems that indicate that both enzymes have comparable spectroscopic, kinetic, thermodynamic, and structural properties.     

Phylogenomic and biochemical characterization of three Legionella pneumophila polypeptide deformylases

Legionella pneumophila is a gram-negative facultative intracellular human pathogen that can cause fatal Legionnaires' disease. Polypeptide deformylase (PDF) is a novel broad-spectrum antibacterial target, and reports of inhibitors of PDF with potent activities against L. pneumophila have been published previously. Here, we report the identification of not one but three putative pdf genes, pdfA, pdfB, and pdfC, in the complete genome sequences of three strains of L. pneumophila. Phylogenetic analysis showed that L. pneumophila PdfA is most closely related to the commonly known gamma-proteobacterial PDFs encoded by the gene def. PdfB and PdfC are more divergent and do not cluster with any specific bacterial or eukaryotic PDF. All three putative pdf genes from L. pneumophila strain Philadelphia 1 have been cloned, and their encoded products have been overexpressed in Escherichia coli and purified. Enzymatic characterization shows that the purified PDFs with Ni2+ substituted are catalytically active and able to remove the N-formyl group from several synthetic polypeptides, although they appear to have different substrate specificities. Surprisingly, while PdfA and PdfB with Zn2+ substituted are much less active than the Ni2+ forms of each enzyme, PdfC with Zn2+ substituted was as active as the Ni2+ form for the fMA substrate and exhibited substrate specificity different from that of Ni2+ PdfC. Furthermore, the catalytic activities of these enzymes are potently inhibited by a known small-molecule PDF inhibitor, BB-3497, which also inhibits the extracellular growth of L. pneumophila. These results indicate that even though L. pneumophila has three PDFs, they can be effectively inhibited by PDF inhibitors which can, therefore, have potent anti-L. pneumophila activity.