Effect of hypoxic episode number and severity on ventilatory long-term facilitation in awake rats.

Episodic hypoxia induces a persistent augmentation of respiratory activity, termed long-term facilitation (LTF). Phrenic LTF saturates in anesthetized animals such that additional episodes of stimulation cause no further increase in LTF magnitude. The present study tested the hypothesis that 1) ventilatory LTF also saturates in awake rats and 2) more severe hypoxia and hypoxic episodes increase the effectiveness of eliciting ventilatory LTF. Minute ventilation was measured in awake, male Sprague-Dawley rats by plethysmography. LTF was elicited by five episodes of 10% O(2) poikilocapnic hypoxia (magnitude: 17.3 +/- 2.8% above baseline, between 15 and 45 min posthypoxia, duration: 45 min) but not 12 or 8% O(2). LTF was also elicited by 10, 20, and 72 episodes of 12% O(2) (19.1 +/- 2.2, 18.9 +/- 1.8, and 19.8 +/- 1.6%; 45, 60, and 75 min, respectively) but not by three or five episodes. These results show that there is a certain range of hypoxia that induces ventilatory LTF and that additional hypoxic episodes may increase the duration but not the magnitude of this response.     

Isolation and culture of rat superior mesenteric artery smooth muscle cells

Summary The structure and function of vascular smooth muscle cells have been extensively investigated with the aid of in vitro culture techniques. The majority of studies have utilized aortic tissue as the source of cells. We present here a method for isolating and culturing smooth muscle cells of the rat superior mesenteric artery, an elasto-muscular vessel that is structurally and functionally different from the aorta. Cells were isolated from partially digested explants and characterized by immunochemical and biochemical techniques. Unlike cultured fibroblasts, the cultured cells stained positive for smooth muscle specific actin. The cells also produced laminin and type IV collagen in culture. This method provides a means for the isolation of large numbers of viable smooth muscle cells from the superior mesenteric artery which can be propagated in culture for in vitro study.     

Going, going, gone: the impact of white-nose syndrome on the summer activity of the little brown bat (Myotis lucifugus)

Since its discovery in the winter of 2005-2006, white-nose syndrome (WNS) has killed over one million little brown bats (Myotis lucifugus) in the American northeast. Although many studies have reported die-offs of bats at winter hibernacula, it is important to understand how bat mortality linked to WNS at winter hibernacula affects bat activity levels in their summer ranges. In the summer (May-August) of 2007, 2008 and 2009, we recorded echolocation calls to determine bat activity at sites along the Hudson River, NY (within approx. 100 km of where WNS was first reported). We documented a 78 per cent decline in the summer activity of M. lucifugus, coinciding with the arrival and spread of WNS. We suggest that mortality of M. lucifugus in winter hibernacula is reflected by reduced levels of activity in the summer and that WNS affects the entire bat population of an area, and not only individual hibernacula.     

Almitrine and doxapram decrease fatigue and increase subsequent recovery in isolated rat diaphragm

McGuire, Michelle, Michael F. Carey, and John J. O'Connor.Almitrine and doxapram decrease fatigue and increase subsequent recovery in isolated rat diaphragm. J. Appl.Physiol. 83(1): 52-58, 1997.The effects ofalmitrine bimesylate and doxapram HCl on isometric force produced by invitro rat diaphragm were studied during direct muscle activation at37°C. Doxapram and almitrine ameliorate respiratory failureclinically by indirectly increasing phrenic nerve activity. This studywas carried out to investigate possible direct actions of these agentson the diaphragm before and after fatigue of the fibers. Two age groupsof animals were chosen [6-14 wk (group1) and 50-55 wk (group2)] because it is known that increasing agedecreases a muscle fiber's resistance to fatigue. Muscle strips wereisolated from both group 1 and group 2 and directly stimulated (2-mspulse duration, 5-15 V) to produce twitch tensions of 1.3 and 2.1 N/cm², respectively. At lowconcentrations, doxapram (20 µg/ml) and almitrine (12 µg/ml)had no effect on twitch contraction or 100-Hz tetanic tension. However,40 µg/ml doxapram and 30 µg/ml almitrine increased twitch tensionby 9.0 ± 1.4 and 11.6 ± 1.9%, respectively, in animals ofgroup 2 (n = 5). A fatigue protocol consistingof low-frequency stimulation (30-Hz trains, 250-ms duration every 2 sfor 5 min) caused a reduction of twitch tension in animals ofgroup 1 (48 ± 4% ofcontrol) and group 2 (28 ± 4% ofcontrol). At 90 min postfatigue, the twitch tension recovered to 72 ± 3 and 42 ± 2% of control values ingroup 1 and group2, respectively. In the presence of doxapram (20 µg/ml), there was a significant increase in the recovery of twitchtension at 90 min in group 1 andgroup 2 (84.5 ± 3.2 and 80.1 ± 2.8%, respectively) compared with controls at 90 min postfatigue. Inthe presence of almitrine (12 µg/ml), there was a full recovery fromfatigue in group 1 animals (100% ofcontrol) and a recovery to 95.6 ± 2.1% of control ingroup 2 animals at 90 min. Theseresults demonstrate a significant improvement in the rapidity andmagnitude of recovery from fatigue in the rat diaphragm muscle in thepresence of both doxapram and, especially, almitrine. These effects maybe due to changes in intracellular calcium, ADP/ATP ratios, or oxygenfree radical scavenging.

     

Hemin interactions and alterations of the subcellular localization of prion protein

Hemin (iron protoporphyrin IX) is a crucial component of many physiological processes acting either as a prosthetic group or as an intracellular messenger. Some unnatural, synthetic porphyrins have potent anti-scrapie activity and can interact with normal prion protein (PrPC). These observations raised the possibility that hemin, as a natural porphyrin, is a physiological ligand for PrPC. Accordingly, we evaluated PrPC interactions with hemin. When hemin (3-10 microM) was added to the medium of cultured cells, clusters of PrPC formed on the cell surface, and the detergent solubility of PrPC decreased. The addition of hemin also induced PrPC internalization and turnover. The ability of hemin to bind directly to PrPC was demonstrated by hemin-agarose affinity chromatography and UV-visible spectroscopy. Multiple hemin molecules bound primarily to the N-terminal third of PrPC, with reduced binding to PrPC lacking residues 34-94. These hemin-PrPC interactions suggest that PrPC may participate in hemin homeostasis, sensing, and/or uptake and that hemin might affect PrPC functions.     

Identification of a frameshift mutation responsible for the silent phenotype of human serum cholinesterase, Gly 117 (GGT----GGAG).

A frameshift mutation that causes a silent phenotype for human serum cholinesterase was identified in the DNA of seven individuals of two unrelated families. The mutation, identified using the polymerase chain reaction, causes a shift in the reading frame from Gly 117, where GGT (Gly)----GGAG (Gly+ 1 base) to a new stop codon created at position 129. This alteration is upstream of the active site (Ser 198), and, if any protein were made, it would represent only 22% of the mature enzyme found in normal serum. Results of analysis of the enzymatic activities in serum agreed with the genotypes inferred from the nucleotide sequence. Rocket immunoelectrophoresis using alpha-naphthyl acetate to detect enzymatic activity showed an absence of cross-reactive material, as expected. One additional individual with a silent phenotype did not show the same frameshift mutation. This was not unexpected, since there must be considerable molecular heterogeneity involved in causes for the silent cholinesterase phenotype. This is the first report of a molecular mechanism underlying the silent phenotype for serum cholinesterase. The analytical approach used was similar to the one we recently employed to identify the mutation that causes the atypical cholinesterase variant.     

Alternative male reproductive tactics in a natural population of prairie voles <Emphasis Type="Italic">Microtus ochrogaster</Emphasis>

Alternative reproductive tactics have been described in male mammals, but little information exists regarding fitness benefits and whether males change tactics. Adult male prairie voles Microtus ochrogaster (Wagner, 1842) display alternative tactics described as resident and wanderer. Enclosure studies provide conflicting data concerning the relative success of each tactic and whether males display one tactic throughout adulthood. To characterize further residents and wanderers in this species, we examined data collected during 5 years of monitoring a natural population in Illinois, USA. We found that during the breeding period, wandering males survived longer, moved longer distances, and were more likely than residents to have scrotal testes. During the nonbreeding period, wandering and resident males differed only in whether or not they established residency. Data on sources and fates of resident and wandering males revealed that a substantial proportion of males switched tactics. Our estimate of the reproductive contribution of wandering males to the population, which is based on the premise that wandering males typically mate with single females, suggests that wanderers contribute 34–38% of young recruited during March through October and 4–12% in November, when single females are less common. Parentage studies in natural populations are necessary to test our estimates.     

Characterization of highly informative cross-species microsatellite panels for the Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) including five novel primers

The Australian dugong (Dugong dugon) and Florida manatee (Trichechus manatus latirostris) are threatened species of aquatic mammals in the order Sirenia. Sirenian conservation and management actions would benefit from a more complete understanding of genetic diversity and population structure. Generally, species-specific microsatellite markers are employed in conservation genetic studies; however, robust markers can be difficult and costly to isolate. To increase the number of available markers, dugong and manatee microsatellite primers were evaluated for cross-species amplification. Furthermore, one manatee and four dugong novel primers are reported. After polymerase chain reaction optimization, 23 (92%) manatee primers successfully amplified dugong DNA, of which 11 (48%) were polymorphic. Of the 32 dugong primers tested, 27 (84%) yielded product in the manatee, of which 17 (63%) were polymorphic. Dugong and manatee primers were compared and the most informative markers were selected to create robust and informative marker-panels for each species. These cross-species microsatellite marker-panels can be employed to assess other sirenian populations and can provide beneficial information for the protection and management of these unique mammals.