David Julian

Birth and fate of proliferative cells in the inner nuclear layer of the mature fish retina

In teleost fish, unlike other vertebrates, the retina continues to grow throughout the animals life both by stretching of the mature tissue and by the addition of new cells. Following larval development, new retinal cell birth is known to occur in a rim at the periphery of the mature retina and in the outer nuclear layer (ONL). We have now found that cell birth and proliferation also occurs in the inner nuclear layer (INL) of the mature fish retina. In rainbow trout (Onchoryncus mykiss), proliferative cells exist in the INL of fish of all ages, at least up to 2 years posthatching. The proliferative cells form clusters in the INL that align in radial columns, reaching from the inner to the outer plexiform layers. The density of proliferative cell clusters changes along the equatorial plane of the retina and is highest near both the nasal and temporal poles. Our data suggest that, after birth, the proliferative cells migrate away from the INL and into the ONL, with a half‐time of about 3 days, and their cell bodies can be seen in the outer plexiform layer. Once they are in the ONL, the proliferative cells continue to divide and likely give rise to the precursor cells that differentiate into new rod photoreceptors. J. Comp. Neurol. 394:271–282, 1998.

Doxorubicin treatment in vivo activates caspase‐12 mediated cardiac apoptosis in both male and female rats

We investigated in vivo the chemotherapeutic anthracycline agents doxorubicin and its ability to activate mitochondrial‐mediated, receptor‐mediated and endoplasmic/sarcoplasmic reticulum‐mediated apoptosis transduction pathways in cardiac tissue from male and female rats. We administered a single low dose of doxorubicin (10 mg/kg of body weight, i.p.) and then isolated mitochondrial and cytosolic proteins one and four days later from the heart. Caspase‐3 protein content and caspase‐3 activity were significantly increased after day four of doxorubicin treatment in both male and female rats. However, while males had DNA fragmentation at day one but not day four following doxorubicin administration, females showed no significant increase in DNA fragmentation at either time. Caspase‐12, localized in the SR, is considered a central caspase, and its activation by cleavage via calpain indicates activation of the SR‐mediated pathway of apoptosis. Cleaved caspase‐12 content and calpain activity significantly increased after day four of doxorubicin treatment in both sexes. In the mitochondrial‐mediated pathway, there were no significant treatment effects observed in cytosolic cytochrome c and cleaved (active) caspase‐9 in either sex. In control rats (saline injection), glutathione peroxidase (GPX) activity and hydrogen peroxide (H2O2) production were lower in females compared to males. Doxorubicin treatment did not significantly affect H2O2, GPX activity or ATP production in isolated mitochondria in either sex. Female rats produced significantly lower levels of H2O2 production one day after doxorubicin treatment, whereas male rats produced significantly less mitochondrial H2O2 four days after doxorubicin treatment. The receptor‐mediated pathway (caspase‐8 and c‐FLIP) showed no evidence of being significantly activated by doxorubicin treatment. Hence, doxorubicin‐induced apoptosis in vivo is mediated by the SR to a greater extent than other apoptotic pathways and should therefore be considered for targeted therapeutic interventions. Moreover, no major sex differences exist in apoptosis signaling transduction cascade due to doxorubicin treatment.

Hydrogen Sulfide Increases Nitric Oxide Production from Endothelial Cells by an Akt-Dependent Mechanism

Hydrogen sulfide (H2S) and nitric oxide (NO) are both gasotransmitters that can elicit synergistic vasodilatory responses in the in the cardiovascular system, but the mechanisms behind this synergy are unclear. In the current study we investigated the molecular mechanisms through which H2S regulates endothelial NO production. Initial studies were performed to establish the temporal and dose-dependent effects of H2S on NO generation using EPR spin trapping techniques. H2S stimulated a twofold increase in NO production from endothelial nitric oxide synthase (eNOS), which was maximal 30 min after exposure to 25–150 μM H2S. Following 30 min H2S exposure, eNOS phosphorylation at Ser 1177 was significantly increased compared to control, consistent with eNOS activation. Pharmacological inhibition of Akt, the kinase responsible for Ser 1177 phosphorylation, attenuated the stimulatory effect of H2S on NO production. Taken together, these data demonstrate that H2S up-regulates NO production from eNOS through an Akt-dependent mechanism. These results implicate H2S in the regulation of NO production in endothelial cells, and suggest that deficiencies in H2S signaling can directly impact processes regulated by NO.

Mitochondrial depolarization following hydrogen sulfide exposure in erythrocytes from a sulfide-tolerant marine invertebrate

SUMMARY Sulfide-tolerant marine invertebrates employ a variety of mechanisms to detoxify sulfide once it has entered their bodies, but their integumentary, respiratory epithelium and circulatory cells may still be exposed to toxic sulfide concentrations. To investigate whether sulfide exposure is toxic to mitochondria of a sulfide-tolerant invertebrate, we used the fluorescent dyes JC-1 and TMRM to determine the effect of sulfide exposure on mitochondrial depolarization in erythrocytes from the annelid Glycera dibranchiata. In erythrocytes exposed to 0.11-1.9 mmol l-1 sulfide for 1 h, the dyes showed fluorescence changes consistent with sulfide-induced mitochondrial depolarization. At the highest sulfide concentration, the extent of depolarization was equivalent to that caused by the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). Even when induced by as little as 0.3 mmol l-1 sulfide, the depolarization was not reversible over a subsequent 5 h recovery period. The mechanism of toxicity was likely not via inhibition of cytochrome c oxidase (COX), since other COX inhibitors and other mitochondrial electron transport chain inhibitors did not produce similar effects. Furthermore, pharmacological inhibition of the mitochondrial permeability transition pore failed to prevent sulfide-induced depolarization. Finally, increased oxidation of the free radical indicators H2DCFDA and MitoSOX™ in erythrocytes exposed to sulfide suggests that sulfide oxidation increased oxidative stress and superoxide production, respectively. Together, these results indicate that sulfide exposure causes mitochondrial depolarization in cells of a sulfide-tolerant annelid, and that this effect, which differs from the actions of other COX inhibitors, may be via increased free radical damage.

Mitotic activation of proliferative cells in the inner nuclear layer of the mature fish retina: regulatory signals and molecular markers.

New neurons continuously differentiate within the otherwise mature retina of teleost fish, both under normal conditions and in response to injury. We investigated the effects of surgical injury and intraocular injection of neurotrophic factors on the mitotic rate of proliferative inner nuclear layer cells (PINC). PINC are continually born in the inner nuclear layer and then migrate to the outer nuclear layer (ONL). Surgical excision of a part of a retina activates PINC mitotic activity near and far from the lesion. In the injured eye, up‐regulation of PINC cells is largest in the dorsonasal sector of the retina, regardless of the site of lesion. Up‐regulation extends even to the unlesioned contralateral eye, where it occurs in the same dorsonasal sector. Intraocular injection of ciliary neurotrophic factor mimics the effect of injury on PINC in the treated eye but not on the untreated contralateral retina. We searched for the expression in PINC of Pax6, a transcription factor linked to retinal progenitor cells and found that less than 0.5% of all PINC cells express it. Importantly, the number of Pax6‐expressing PINC does not change significantly in the retinas subjected to any of the experimental manipulations tested. Under normal conditions, the default fate of PINC cells is to migrate to the ONL and, likely, replenish the rod progenitor pool. PINC respond to injury (both surgical and light‐dependent) by increasing their mitotic rate; this increase is long lived, but there are no changes in the expression level of Pax6. PINC probably are a heterogenous cell population that can be specified for ultimate, different purposes: creating rod precursors, creating founder cells, creating cone precursors. Several fates are recognized now, but others may also be possible. J. Comp. Neurol. 451:127–141, 2002.

Sulfide permeability in the marine invertebrate Urechis caupo

SummaryHydrogen sulfide can reach toxic concentrations in the burrow-water of the echiuran worm Urechis caupo during low tide. Its two large epithelial surfaces, the thick muscular body wall and the thin-walled hindgut are in constant contact with the environment. Hindgut inflation of up to 2 ml water·g wet weight-1 causes tissue stretch. To determine if these body surfaces present a barrier to sulfide influx, the total permeability coefficient PT was measured at different degrees of stretch in diffusion chambers at pH 6.0, 7.0, and 8.0, and specific permeability coefficients PH2S and PHS- were calculated. Both the body wall and the hindgut were more permeable to H2S than HS-. The body wall showed no significant increase in sulfide permeability with natural degrees of stretch, and the mean PH2S and PHS- were 0.17 and 0.063 cm·h-1, respectively. The sulfide permeability of the hindgut was increased by stretch, with the relative permeability of H2S increasing faster than that of HS-. Unstretched hindgut mean PH2S and PHS- were 0.095 and 0.11 cm·h-1, respectively, and stretched hindgut mean PH2S and PHS- were 1.8 and 0.16 cm·h-1, respectively. A model of sulfide influx in the natural environment indicates that even if the hindgut is kept uninflated, the coelomic fluid of U. caupo would have toxic sulfide concentrations well before the end of a 2-h tidal exposure in the absence of a sulfide elimination mechanism.

Oxygen and steroid concentrations in preovulatory follicles of lactating dairy cows exposed to acute heat stress.

Maternal heat stress reduces oocyte competence for fertilization and post-fertilization development, but the mechanism is unknown. The present experiment investigated two potential mechanisms: (1) reduced oxygen delivery to the preovulatory follicle (due to increased thermoregulatory vascular perfusion of skin and respiratory tract); (2) reduced follicular steroid synthesis. These hypotheses were tested by measuring the fractional concentration of oxygen and concentrations of estradiol-17beta and progesterone in follicular fluid of the preovulatory follicle of lactating Holstein cows. Estrous cycles were synchronized using GnRH on Day -9 and PGF(2alpha) on Day -2. On Day 0, all cows without a CL and with a large preovulatory follicle were assigned to control or heat stress treatments for 1d (beginning at 1030 h). Between 4 and 6 h after treatment (1430-1630 h), follicular fluid was aspirated by transvaginal puncture, and fractional oxygen concentration in follicular fluid of the dominant follicle was determined with a fluorometric fiber-optic oxygen sensor. There was no significant effect of heat stress on follicular fluid P(O2) or concentrations of estradiol-17beta or progesterone among cows that had follicular fluid steroid concentrations considered typical of a preovulatory follicle. Follicular oxygen concentration was 6.9+/-0.4% for control cows and 7.3+/-0.3% for heat-stressed cows. Oxygen concentration tended to be inversely correlated to follicular diameter (P=0.09). In conclusion, it was unlikely that reduced oocyte competence due to acute heat stress was caused by reductions in follicular concentrations of oxygen, estradiol-17beta, or progesterone.

Hydrogen Sulfide Induces Oxidative Damage to RNA and DNA in a Sulfide-Tolerant Marine Invertebrate

Hydrogen sulfide acts as an environmental toxin across a range of concentrations and as a cellular signaling molecule at very low concentrations. Despite its toxicity, many animals, including the mudflat polychaete Glycera dibranchiata, are periodically or continuously exposed to sulfide in their environment. We tested the hypothesis that a broad range of ecologically relevant sulfide concentrations induces oxidative stress and oxidative damage to RNA and DNA in G. dibranchiata. Coelomocytes exposed in vitro to sulfide (0–3 mmol L−1 for 1 h) showed dose‐dependent increases in oxidative stress (as 2′,7′‐dichlorofluorescein fluorescence) and superoxide production (as dihydroethidine fluorescence). Coelomocytes exposed in vitro to sulfide (up to 0.73 mmol L−1 for 2 h) also acquired increased oxidative damage to RNA (detected as 8‐oxo‐7,8‐dihydroguanosine) and DNA (detected as 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine). Worms exposed in vivo to sulfide (0–10 mmol L−1 for 24 h) acquired elevated oxidative damage to RNA and DNA in both coelomocytes and body wall tissue. While the consequences of RNA and DNA oxidative damage are poorly understood, oxidatively damaged deoxyguanosine bases preferentially bind thymine, causing G‐T transversions and potentially causing heritable point mutations. This suggests that sulfide can be an environmental mutagen in sulfide‐tolerant invertebrates.

Thiosulfate elimination and permeability in a sulfide-adapted marine invertebrate.

Oxidation of hydrogen sulfide to thiosulfate is one of the best‐characterized mechanisms by which animals adapted to sulfide minimize its toxicity, but the mechanism of thiosulfate elimination in these animals has remained unclear. In this study, we examined the accumulation and elimination of thiosulfate in the sulfide‐adapted marine worm Urechis caupo. The coelomic fluid of U. caupo exposed to 50–100 μmol L−1 sulfide in hypoxic seawater (Po2 ca. 10 kPa) accumulated ( \documentclass{aastex} \usepackage{amsbsy} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{bm} \usepackage{mathrsfs} \usepackage{pifont} \usepackage{stmaryrd} \usepackage{textcomp} \usepackage{portland,xspace} \usepackage{amsmath,amsxtra} \usepackage[OT2,OT1]{fontenc} \newcommand\cyr{ \renewcommand\rmdefault{wncyr} \renewcommand\sfdefault{wncyss} \renewcommand\encodingdefault{OT2} \normalfont \selectfont} \DeclareTextFontCommand{\textcyr}{\cyr} \pagestyle{empty} \DeclareMathSizes{10}{9}{7}{6} \begin{document} \landscape