Cardioprotective effects and pharmacokinetic properties of a controlled release formulation of a novel hydrogen sulfide donor in rats with acute myocardial infarction

We previously reported that S-propargyl-cysteine (SPRC) exerts cardioprotective effects by elevating H₂S levels via the CSE/H₂S pathway. In the present study, we investigated the cardioprotective effects and pharmacokinetic properties of a controlled release formulation of SPRC (CR-SPRC) in an in vivo rat model of myocardial infarction (MI). Rats were randomly assigned to seven groups that were pre-treated with CR-SPRC daily for 7 days prior to ligation of the left anterior descending coronary artery to induce MI. Cardiac function and infarct size were determined after MI, and we examined the activity of antioxidant enzymes, expression of anti-inflammation proteins and hydrogen sulfide levels. Mixed-mode, reversed-phase and cation-exchange HPLC–MS/MS were used to compare the pharmacokinetic properties of CR-SPRC and SPRC. CR-SPRC significantly reduced infarct size and creatine kinase (CK) and lactate dehydrogenase (LDH) leakage and it preserved cardiac function during MI. CR-SPRC displayed antioxidant properties, preserving glutathione (GSH), catalase (CAT) and superoxide dismutase (SOD) levels whereas reducing malondialdehyde (MDA) levels. Moreover, CR-SPRC significantly reduced the protein levels of inflammatory biomarkers (phospho-NF-κB p65/NF-κB p65, TNF-α) and increased cystathionine-γ-lyase (CSE) and Iκ-Bα protein levels. CR-SPRC had better pharmacokinetic properties than SPRC, with a reduced concentration peak (C_max), prolonged time to reach peak concentration (T_max), prolonged mean residence time (MRT_inf) and increased AUC_0–t. CR-SPRC showed protective effects against MI via the CSE/H₂S pathway and demonstrated better cardioprotective effects than SPRC by prolonging the release of endogenous H₂S.     

Leukocyte trafficking in alveoli and airway passages

Many pulmonary diseases preferentially affect the large airways or the alveoli. Although the mechanisms are often particular to each disease process, site-specific differences in leukocyte trafficking and the regulation of inflammation also occur. Differences in the process of margination, sequestration, adhesion, and migration occur that can be attributed to differences in anatomy, hemodynamics, and the expression of proteins. The large airways are nourished by the bronchial circulation, whereas the pulmonary circulation feeds the distal lung parenchyma. The presence of different cell types in large airways from those in alveoli might contribute to site-specific differences in the molecular regulation of the inflammatory process.     

Respiratory responses of the air-breathing fish Hoplosternum littorale to hypoxia and hydrogen sulfide

The present study analyzes the respiratory responses of the neotropical air-breathing fish Hoplosternum littorale to graded hypoxia and increased sulfide concentrations. The oxygen uptake (VO2), critical O2 tension (PcO2), respiratory (fR) and air-breathing (fRA) frequencies in response to graded hypoxia were determined for fish acclimated to 28 degrees C. H. littorale was able to maintain a constant VO2 down to a PcO2 of 50 mm Hg, below which fish became dependent on the environmental O2 even with significant increases in fR. The fRA was kept constant around 1 breath h(-1) above 50 mm Hg and increased significantly below 40 mm Hg, reaching maximum values (about 4.5 breaths h(-1)) at 10 mm Hg. The lethality to sulfide concentrations under normoxic and hypoxic conditions were also determined along with the fRA. For the normoxic fish the sulfide lethal limit was about 70 microM, while in the hypoxic ones this limit increased to 87 muM. The high sulfide tolerance of H. littorale may be attributed to the air-breathing capability, which is stimulated by this compound.     

A novel hydrogen sulfide donor causes stomatal opening and reduces nitric oxide accumulation

Effects of hydrogen sulfide (H₂S) on plant physiology have been previously studied, but such studies have relied on the use of NaSH as a method for supplying H₂S to tissues. Now new compounds which give a less severe H₂S shock and a more prolonged exposure to H₂S have been developed. Here the effects of one such compound, GYY4137, has been investigated to determine its effects on stomatal closure in Arabidopsis thaliana. It was found that both NaSH and GYY4137 caused stomatal opening in the light and prevented stomatal closure in the dark. Nitric oxide (NO) has been well established as a mediator of stomatal movements and here it was found that both NaSH and GYY4137 reduced the accumulation of NO in guard cells, perhaps suggesting a mode of action for H₂S in this system. GYY4137, and future related compounds, will be important tools to unravel the effects of plant exposure to H₂S and to determine how H₂S may fit into plant cell signalling pathways.     

Development of a method to measure hydrogen sulfide in wine fermentation

A hydrogen sulfide (H2S) detecting tube was developed for the quantitative determination of H2S produced by yeast during laboratory scale wine fermentations. The detecting tube consisted of a small transparent plastic tube packed with an H2S-sensitive color-indicating medium. The packed medium changed color, with the color change progressing upward from the bottom of the tube, upon exposure to H2S produced by yeast during fermentation. A calibration study using a standard H2S gas showed that the length of the portion that darkened was directly related to the quantity of H2S (microg) with a high correlation coefficient (r2=0.9997). The reproducibility of the H2S detecting tubes was determined with five repetitive measurements using a standard H2S solution [5.6 microg/200 ml (28 ppb)], which resulted in a coefficient of variation of 3.6% at this level of H2S. With the sulfide detecting tubes, the production of H2S was continuously monitored and quantified from laboratory scale wine fermentations with different yeast strains and with the addition of different levels of elemental sulfur to the grape juice. This sulfide detecting tube technology may allow winemakers to quantitatively measure H2S produced under different fermentation conditions, which will eventually lead winemakers to better understand the specific factors and conditions for the excessive production of H2S during wine fermentation in a large production scale.     

The response of Caenorhabditis elegans to hydrogen sulfide and hydrogen cyanide

Hydrogen sulfide (H2S), an endogenously produced small molecule, protects animals from various stresses. Recent studies demonstrate that animals exposed to H2S are long lived, resistant to hypoxia, and resistant to ischemia-reperfusion injury. We performed a forward genetic screen to gain insights into the molecular mechanisms Caenorhabditis elegans uses to appropriately respond to H2S. At least two distinct pathways appear to be important for this response, including the H2S-oxidation pathway and the hydrogen cyanide (HCN)-assimilation pathway. The H2S-oxidation pathway requires two distinct enzymes important for the oxidation of H2S: the sulfide:quinone reductase sqrd-1 and the dioxygenase ethe-1. The HCN-assimilation pathway requires the cysteine synthase homologs cysl-1 and cysl-2. A low dose of either H2S or HCN can activate hypoxia-inducible factor 1 (HIF-1), which is required for C. elegans to respond to either gas. sqrd-1 and cysl-2 represent the entry points in the H2S-oxidation and HCN-assimilation pathways, respectively, and expression of both of these enzymes is highly induced by HIF-1 in response to both H2S and HCN. In addition to their role in appropriately responding to H2S and HCN, we found that cysl-1 and cysl-2 are both essential mediators of innate immunity against fast paralytic killing by Pseudomonas. Furthermore, in agreement with these data, we showed that growing worms in the presence of H2S is sufficient to confer resistance to Pseudomonas fast paralytic killing. Our results suggest the hypoxia-independent hif-1 response in C. elegans evolved to respond to the naturally occurring small molecules H2S and HCN.     

The slow-releasing hydrogen sulfide donor, GYY4137, exhibits novel anti-cancer effects in vitro and in vivo

The slow-releasing hydrogen sulfide (H₂S) donor, GYY4137, caused concentration-dependent killing of seven different human cancer cell lines (HeLa, HCT-116, Hep G2, HL-60, MCF-7, MV4-11 and U2OS) but did not affect survival of normal human lung fibroblasts (IMR90, WI-38) as determined by trypan blue exclusion. Sodium hydrosulfide (NaHS) was less potent and not active in all cell lines. A structural analogue of GYY4137 (ZYJ1122) lacking sulfur and thence not able to release H₂S was inactive. Similar results were obtained using a clonogenic assay. Incubation of GYY4137 (400 µM) in culture medium led to the generation of low (<20 µM) concentrations of H₂S sustained over 7 days. In contrast, incubation of NaHS (400 µM) in the same way led to much higher (up to 400 µM) concentrations of H₂S which persisted for only 1 hour. Mechanistic studies revealed that GYY4137 (400 µM) incubated for 5 days with MCF-7 but not IMR90 cells caused the generation of cleaved PARP and cleaved caspase 9, indicative of a pro-apoptotic effect. GYY4137 (but not ZYJ1122) also caused partial G₂/M arrest of these cells. Mice xenograft studies using HL-60 and MV4-11 cells showed that GYY4137 (100–300 mg/kg/day for 14 days) significantly reduced tumor growth. We conclude that GYY4137 exhibits anti-cancer activity by releasing H₂S over a period of days. We also propose that a combination of apoptosis and cell cycle arrest contributes to this effect and that H₂S donors should be investigated further as potential anti-cancer agents.     

Eukaryotic and prokaryotic contributions to colonic hydrogen sulfide synthesis

Hydrogen sulfide (H(2)S) is an important modulator of many aspects of digestive function, both in health and disease. Colonic tissue H(2)S synthesis increases markedly during injury and inflammation and appears to contribute to resolution. Some of the bacteria residing in the colon can also produce H(2)S. The extent to which bacterial H(2)S synthesis contributes to what is measured as colonic H(2)S synthesis is not clear. Using conventional and germ-free mice, we have delineated the eukaryotic vs. prokaryotic contributions to colonic H(2)S synthesis, both in healthy and colitic mice. Colonic tissue H(2)S production is entirely dependent on the presence of the cofactor pyridoxal 5'-phosphate (vitamin B(6)), while bacterial H(2)S synthesis appears to occur independent of this cofactor. As expected, approximately one-half of the H(2)S produced by feces is derived from eukaryotic cells. While colonic H(2)S synthesis is markedly increased when the tissue is inflamed, and, in proportion to the extent of inflammation, fecal H(2)S synthesis does not change and tissue granulocytes do not appear to be the source of the elevated H(2)S production. Rats fed a B vitamin-deficient diet for 6 wk exhibited significantly diminished colonic H(2)S synthesis, but fecal H(2)S synthesis was not different from that of rats on the control diet. Our results demonstrate that H(2)S production by colonic bacteria does not contribute significantly to what is measured as colonic tissue H(2)S production, using the acetate trapping assay system employed in this study.     

Leukocyte trafficking and myocardial reperfusion injury in ICAM-1/P-selectin-knockout mice

P-selectin and intercellular adhesion molecule-1 (ICAM-1) mediate early interaction and adhesion of neutrophils to coronary endothelial cells and myocytes after myocardial ischemia and reperfusion. In the present study, we examined the physiological consequences of genetic deletions of ICAM-1 and P-selectin in mice. In wild-type mice, after 1 h of ischemia followed by reperfusion, neutrophil influx into the area of ischemia was increased by 3 h with a peak at 24 h and a decline by 72 h. ICAM-1/P-selectin-deficient mice showed a significant reduction in neutrophils by immunohistochemistry or by myeloperoxidase activity at 24 h but no significant difference at 3 h. Infarct size (area of necrosis/area at risk) assessed 24 h after reperfusion was not different between wild-type and deficient mice after 30 min and 1 h of occlusion. Mice with a deficiency in both ICAM-1 and P-selectin have impaired neutrophil trafficking without a difference in infarct size due to myocardial ischemia-reperfusion.     

Ozone and hydrogen peroxide as strategies to control biomass in a trickling filter to treat methanol and hydrogen sulfide under acidic conditions

Applied Microbiology and Biotechnology - The operation and performance of a biotrickling filter for methanol (MeOH) and hydrogen sulfide (H2S) removal at acid pH was studied. Excess biomass in the...     

Mitochondrial adaptations to utilize hydrogen sulfide for energy and signaling

Sulfur is a versatile molecule with oxidation states ranging from -2 to +6. From the beginning, sulfur has been inexorably entwined with the evolution of organisms. Reduced sulfur, prevalent in the prebiotic Earth and supplied from interstellar sources, was an integral component of early life as it could provide energy through oxidization, even in a weakly oxidizing environment, and it spontaneously reacted with iron to form iron-sulfur clusters that became the earliest biological catalysts and structural components of cells. The ability to cycle sulfur between reduced and oxidized states may have been key in the great endosymbiotic event that incorporated a sulfide-oxidizing α-protobacteria into a host sulfide-reducing Archea, resulting in the eukaryotic cell. As eukaryotes slowly adapted from a sulfidic and anoxic (euxinic) world to one that was highly oxidizing, numerous mechanisms developed to deal with increasing oxidants; namely, oxygen, and decreasing sulfide. Because there is rarely any reduced sulfur in the present-day environment, sulfur was historically ignored by biologists, except for an occasional report of sulfide toxicity. Twenty-five years ago, it became evident that the organisms in sulfide-rich environments could synthesize ATP from sulfide, 10?years later came the realization that animals might use sulfide as a signaling molecule, and only within the last 4?years did it become apparent that even mammals could derive energy from sulfide generated in the gastrointestinal tract. It has also become evident that, even in the present-day oxic environment, cells can exploit the redox chemistry of sulfide, most notably as a physiological transducer of oxygen availability. This review will examine how the legacy of sulfide metabolism has shaped natural selection and how some of these ancient biochemical pathways are still employed by modern-day eukaryotes.     

Evaluation of a microbial method to reduce hydrogen sulfide levels in a porous rock biofilm

Summary The efficacy of nitrate addition, with and without inoculation with a sulfide-resistant strain ofThiobacillus denitrificans (strain F), in reducing sulfide levels in an experimental system using cores and subsurface formation water from a gas storage facility was examined. The addition of nitrate (40 mM) alone to the formation water injected into core systems operated at hydraulic retention times of 3.2 and 16.7 h resulted in lower effluent sulfide concentrations, from an influent concentration of about 170–190 M to an effluent concentration of 110 and 3 M, respectively. A reduction in effluent nitrate concentrations in both core systems indicated the presence of indigenous nitrate-using populations. After strain F was inoculated into the core system operated at the shorter retention time, the effluent sulfide concentration decreased from 110 to 16–25 M. The effluent sulfate concentration increased, and the effluent nitrate concentration decreased concomitant with the presence of high concentrations of denitrifying thiobacilli in the inoculated core system. The denitrifying thiobacilli detected after inoculation were presumed to be strain F since these organisms were not detected in this core system before inoculation, or in any of the samples from the uninoculated core system. These data suggest that the efficacy of the nitrate treatment may depend on the residence times of the liquids in the core system, and that inoculation with strain F was required to reduce sulfide levels to <20 M in the core system operated at a short hydraulic retention time.     

内、外源性硫化氢在脂多糖所致大鼠急性肺损伤中的作用

目的：探讨内、外源性硫化氢（H2S）在脂多糖（LPS）所致大鼠急性肺损伤（ALI）中的作用并初探其机制。方法：将120只SD大鼠随机分为对照组、IPS组（经气管内滴注LPS复制ALI模型）、NaHS＋LPS组和炔丙基甘氨酸（PPG）＋LPS组。给药后4h或8h处死动物,测定肺系数;光镜观察肺组织形态学改变;化学法检测血浆H2S、NO和CO含量、肺组织丙二醛（MDA）含量、胱硫醚-γ-裂解酶（CSE）、诱导型一氧化氮合酶（iNOS）和血红素加氧酶（HO）活性以及支气管肺泡灌洗液（BALF）中中性粒细胞（PMN）数目和蛋白含量的变化;用免疫组织化学法检测肺组织iNOS、HO-1蛋白表达。再将血浆H2S含量与上述指标进行相关性分析。结果：气管内滴注LPS可引起肺组织明显的形态学改变;肺系数和肺组织MDA含量增加;BALF中PMN数目和蛋白含量增加;血浆H2S含量和肺组织CSE活性下降;肺组织iNOS活性、HO活性和iNOS蛋白表达、HO-1蛋白表达增强,血浆NO含量、CO含量增加。预先给予NaHS可显著减轻LPS所致上述指标的改变;而预先给予PIG可加重LPS所致肺损伤,使BALF中PMN数目和蛋白含量、血浆NO含量、肺组织iNOS活性和iNOS蛋白表达进一步增加,但对血浆CO含量、肺组织HO活性和HO-1蛋白表达无明显影响。HS含量与CSE活性、血浆CO含量、肺组织HO-1活性呈正相关（r值=0．945—0．987,P均〈0．01）;与其他指标呈负相关（r值=-0．994～-0．943,P均〈0．01）。结论：H2S／CSE体系的下调在LPS所致大鼠Ⅲ的发病学中有一定作用,内、外源性H2S具有抗LPS所致Au的作用,该作用可能与其抗氧化效应、减轻PMN所致肺过度的炎症反应以及下调NO／iNOS体系、上调CO／HO—1体系有一定关系。     

Temporal effects of estrogen and progesterone on behavioral and endocrinological responses to acute cocaine administration.

Estrogen and progesterone have been postulated to play a key role modulating cocaine-induced behavioral and neurochemical activation in female rats. This study investigated the temporal relationship between estrogen and progesterone in the modulation of cocaine-induced behavioral alterations. Ovariectomized Fischer rats received s.c. injections of estradiol benzoate 48 hr prior to cocaine or saline treatment and one s.c. injection of progesterone concurrently or 1, 4, 20, 24, 30, 44 or 48 hr after estrogen treatment. Forty-eight hours after estrogen treatment rats received either a single i.p. injection of 15 mg/kg of cocaine or 0.9% saline. Overall, cocaine induced increases in locomotor behaviors (ambulatory and rearing activity). A bimodal interaction between estrogen and progesterone was observed in the modulation of all locomotor activities. A gradual increase in behaviors, which peaked when progesterone was administered 24 hr after estrogen was followed by an inhibition of both ambulatory and rearing activity when progesterone was administered for a shorter period of time. This estrogen and progesterone interaction was not observed in the modulation of cocaine-induced stereotypic activity. However, shorter administration of progesterone in relation to estrogen administration resulted in lowered benzoylecgonine plasma levels when compared to longer progesterone administration times. On the other hand, longer administration of progesterone (48 hr of estrogen and progesterone) caused increases in corticosterone levels in cocaine-treated rats. Thus, the temporal interaction between estrogen and progesterone in the regulation of cocaine metabolism and hypothalamic-pituitary-axis (HPA) activation do not completely correlate with that observed for locomotor behavioral activation. Taken together, these results suggest that temporal interactions between estrogen and progesterone may underlie some of the previously reported estrous cycle and sex effects on cocaine-induced behavioral and endocrinological alteration.     

Leukocyte migratory responses to apoptosis: The attraction and the distraction

An expanding body of evidence demonstrates that cells undergoing apoptosis send out a selection of molecular navigational signals including proteins, lipids and nucleotides that serve to recruit phagocytes to the dying targets, which are subsequently engulfed and removed. This homeostatic process is essentially non-phlogistic, contrasting markedly with the acute inflammatory responses elicited in phagocytes by damaging or infectious agents. The “professional” scavengers of apoptotic cells are mononuclear phagocytes—the macrophages—and sites of high-rate apoptosis are clearly characterized by macrophages associated with the apoptotic cells. By contrast, members of the other class of professional phagocytes—the granulocytes—are not recruited to sites of apoptosis as a direct consequence of the cell-death program. Indeed, recent work indicates that apoptotic cells release a mixture of migratory cues to leukocytes in order to selectively attract mononuclear phagocytes but not granulocytes through functional balancing of positive and negative signals. Here we discuss these molecular mechanisms that not only serve as migratory cues but also may activate responding phagocytes to engulf apoptotic cells effectively. Finally, we speculate upon new therapeutic opportunities these mechanisms offer for a range of pathological conditions, including inflammatory disorders and cancer.Key words: apoptosis, migration, chemotaxis, macrophage, monocyte, granulocyte, phagocytosis, lactoferrin, ATP, fractalkine     

硫化氢对急性心肌缺血大鼠心肌线粒体损伤的影响

目的：探讨硫化氢（H2S）对急性心肌缺血大鼠线粒体功能的影响,并探讨其改善急性心肌缺血损伤的作用机制。方法：通过结扎大鼠左冠状动脉前降支建立急性心肌缺血模型。雄性SD大鼠48只随机分为6组（n=8）：假手术组,缺血组,缺血＋硫氢化钠（NaHS）低、中、高剂量组和缺血＋炔丙基甘氨酸（PPG）组。透射电镜观察心肌组织线粒体超微结构;检测血浆中H2S含量、心肌组织CSE活性;测定心肌线粒体活力、膜肿胀度及线粒体总ATP酶、谷胱甘肽过氧化物酶（GSH-PX）、超氧化物歧化酶（SOD）的活性和丙二醛（MDA）含量。结果：与假手术组比较,缺血组大鼠血浆H2S含量和心肌组织中CSE活性降低;心肌线粒体膜肿胀,线粒体活力下降;线粒体中MDA含量明显升高,ATP酶、SOD、GSH-Px活性明显降低（P〈0.01）。与缺血组比较,缺血＋NaHS低、中、高剂量组大鼠血浆H2S含量和心组织中CSE活性均升高;缺血＋NaHS中、高剂量组大鼠心肌线粒体MDA含量明显减少,膜肿胀度减轻;缺血＋NaHS低、中、高剂量组线粒体活力有所恢复,ATP酶、SOD、GSH-Px的活性明显升高（P〈0.05或P〈0.01）。PPG可部分减弱H2S的心肌保护作用（P〈0.05或P〈0.01）。结论：H2S可增强线粒体ATP酶、SOD、GSH-Px的活性,降低线粒体脂质过氧化水平,从而起到对大鼠急性心肌缺血的保护作用。     

Emersion in the mangrove forest fishRivulus marmoratus: a unique response to hydrogen sulfide

Synopsis The mangrove forest fishRivulus marmoratus (Cyprinodontidae) has frequently been observed out of water, a phenomenon generally attributed to habitat drying. We tested the hypothesis that hydrogen sulfide, a substance characteristically found in their environment, can serve as a stimulus for emersion. In the field we foundR. marmoratus in water with low to moderate levels (<250 ppb) of HS. In the laboratory,R. marmoratus leaped from water contaminated with H₂S at ecologically relevant concentrations (median response at 123 ppb). Aquatic hypoxia did not induce emersion, but prey capture did. Oxygen consumption by both juveniles and adults decreased significantly in air (27 and 25%, respectively). Our results suggest that avoidance of H₂S and the ability to survive terrestrial conditions enable this species to permanently occupy an area of the forest unavailable to other fishes. Furthermore, because a variety of stimuli lead to etriersion inR. marmoratus, terrestriality in this species is likely a generalized response to environmental stress as well as a means of exploiting terrestrial resources.     

Precursors and inhibitors of hydrogen sulfide synthesis affect acute hypoxic pulmonary vasoconstriction in the intact lung

The effects of hydrogen sulfide (H(2)S) and acute hypoxia are similar in isolated pulmonary arteries from various species. However, the involvement of H(2)S in hypoxic pulmonary vasoconstriction (HPV) has not been studied in the intact lung. The present study used an intact, isolated, perfused rat lung preparation to examine whether adding compounds essential to H(2)S synthesis or to its inhibition would result in a corresponding increase or decrease in the magnitude of HPV. Western blots performed in lung tissue identified the presence of the H(2)S-synthesizing enzymes, cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfur transferase (3-MST), but not cystathionine β-synthase (CBS). Adding three H(2)S synthesis precursors, cysteine and oxidized or reduced glutathione, to the perfusate significantly increased peak arterial pressure during hypoxia compared with control (P < 0.05). Adding α-ketoglutarate to enhance the 3-MST enzyme pathway also resulted in an increase (P < 0.05). Both aspartate, which inhibits the 3-MST synthesis pathway, and propargylglycine (PPG), which inhibits the CSE pathway, significantly reduced the increases in arterial pressure during hypoxia. Diethylmaleate (DEM), which conjugates sulfhydryls, also reduced the peak hypoxic arterial pressure at concentrations >2 mM. Finally, H(2)S concentrations as measured with a specially designed polarographic electrode decreased markedly in lung tissue homogenate and in small pulmonary arteries when air was added to the hypoxic environment of the measurement chamber. The results of this study provide evidence that the rate of H(2)S synthesis plays a role in the magnitude of acute HPV in the isolated perfused rat lung.     

Cortisol and behavioral responses to enrichment in military working dogs

Our objective was to compare the effects of two schedules of enrichment exposure, a distributed (high frequency) and regular regime versus a mass (low frequency) and irregular regime, on the cortisol and behavioral responses of 14 kennelled military working dogs. The enrichment experiences were exercise coupled with human contact. Both groups of dogs were exposed to approximately the same total amount of enrichment per week for a total of 7 weeks. The distributed and regular regime (DRR) furnished enrichment for 20-min intervals three to four times a day every weekday; the mass and irregular regime (MIR) randomly furnished enrichment for 16 or 24 continuous hours 1 day out of 5. Our data show that: (1) cortisol concentrations of DRR dogs significantly decreased between the first and the last week of observations; this was not the case in MIR dogs; (2) at the 7th week DRR dogs had lower cortisol concentrations than did MIR dogs; (3) all dogs in each group exhibited stereotypies. Our results suggest that MIR dogs underwent a decline in welfare throughout the experiment, whereas DRR dogs appeared to undergo a period of acute stress at the beginning of their training that resolved over 7 weeks. This study suggests that the welfare of kennelled dogs, particularly military working dogs, is affected among other factors by schedules of exercise and contact with people.     

Biogeographic variation in behavioral and morphological responses to predation risk

The expression of prey antipredator defenses is often related to ambient consumer pressure, and prey express greater defenses under intense consumer pressure. Predation is generally greater at lower latitudes, and antipredator defenses often display a biogeographic pattern. Predation pressure may also vary significantly between habitats within latitudes, making biogeographic patterns difficult to distinguish. Furthermore, invasive predators may also influence the expression of prey defenses in ecological time. The purpose of this study was to determine how these factors influence the strength of antipredator responses. To assess patterns in prey antipredator defenses based upon geographic range (north vs. south), habitat type (wave-protected vs. wave-exposed shores), and invasive predators, we examined how native rock (Cancer irroratus) and invasive green (Carcinus maenas) crab predators influence the behavioral and morphological defenses of dogwhelk (Nucella lapillus) prey from habitats that differ in wave exposure across an ~230 km range within the Gulf of Maine. The expression of behavioral and morphological antipredatory responses varied according to wave exposure, geographic location, and predator species. Dogwhelks from areas with an established history with green crabs exhibited the largest behavioral and morphological antipredator responses to green crabs. Dogwhelk behavioral responses to rock crabs did not vary between habitats or geographic regions, although morphological responses were greater further south where predation pressure was greatest. These findings suggest that dogwhelk responses to invasive and native predators vary according to geographic location and habitat, and are strongly affected by ambient predation pressure due to the invasion history of an exotic predator.