bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytokines and growth factors in keratinocytes and sweat glands in chronic venous leg ulcers. An immunohistochemical study
Authors: Tian Yan‐Wu , Stacey Michael C.
Published: August 2003

The role of growth factors and cytokines in the impaired healing of chronic leg ulcers remains uncertain. The aim of this study was to determine whether changes in the amount and location of cytokines and growth factors may be associated with impaired healing in chronic leg ulcers. Biopsies from leg ulcers of 21 patients and from normal skin of nine healthy volunteers were examined immunohistochemically for selected growth factors and cytokines. Greater staining intensity was found in keratinocytes at the edges of ulcers compared to normal skin, or skin adjacent to the ulcers. Staining at the ulcer edge was more intense in nonhealing ulcers for only vascular endothelial growth factor and platelet‐derived growth factor, whereas staining in the adjacent skin was more intense for all factors in the nonhealing phase. For all factors staining was cytoplasmic, suggesting production in these areas. This study shows up‐regulation of the production of cytokines and growth factors in keratinocytes of chronic leg ulcers that is greater when the ulcers are nonhealing. (WOUND REP REG 2003;11:316–325)
The study of cytokine dynamics at the operation site after mastectomy
Authors: Chow Louis W. C. , Loo Wings T. Y. , Yuen Kwok‐Yung , Cheng Carter
Published: August 2003

Twenty‐nine patients, who each received modified radical mastectomy, were recruited for this study. Wounds were inspected daily for the presence of flap necrosis, infection, and seroma. Drain fluid (20 ml) was collected at 6 a.m. on postoperative days 1, 2, and 5 and the levels of interleukin (IL)‐4, IL‐6, tumor necrosis factor‐α, and interferon‐γ determined. For patients with no wound events, only IL‐6 levels were elevated during the initial phase, but in the later phase the IL‐6 levels dropped with a corresponding rise in tumor necrosis factor‐α levels. In patients with flap necrosis, there was a sequential rise of IL‐4 on day 1, IL‐6 on day 2, and tumor necrosis factor‐α on day 5, but only IL‐4 was found to be a statistically significant factor associated with necrosis. In patients with seroma, the levels of IL‐4 and interferon‐γ were persistently low and were both statistically significant. To conclude, IL‐6 and tumor necrosis factor‐α are important in normal postoperative wound healing and IL‐4 and interferon‐γ may be associated with postoperative necrosis and seroma. (WOUND REP REG 2003;11:326–330)
In vitro mechanical compression induces apoptosis and regulates cytokines release in hypertrophic scars
Authors: Renò Filippo , Sabbatini Maurizio , Lombardi Francesca , Stella Maurizio , Pezzuto Carla , Magliacani Gilberto , Cannas Mario
Published: August 2003

Hypertrophic scars resulting from severe burns are usually treated by continuous elastic compression. Although pressure therapy reaches success rates of 60–85% its mechanisms of action are still poorly understood. In this study, apoptosis induction and release of interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α) were evaluated in normal (n = 3) and hypertrophic (=7) scars from burns after in vitro mechanical compression. In the absence of compression (basal condition) apoptotic cells, scored using terminal deoxyribonucleotidyl transferase assay, were present after 24 hours in the derma of both normal scar (23 ± 0.4% of total cell) and hypertrophic scar (11.3 ± 1.4%). Mechanical compression (constant pressure of 35 mmHg for 24 hours) increased apoptotic cell percentage both in normal scar (29.5 ± 0.4%) and hypertrophic scar (29 ± 1.7%). IL‐1β released in the medium was undetectable in normal scar under basal conditions while in hypertrophic scar the IL‐1β concentration was 3.48 ± 0.2 ng/g. Compression in hypertrophic scar‐induced secretion of IL‐1β twofold higher compared to basal condition. (7.72 ± 0.2 ng/g). TNF‐α basal concentration measured in normal scar medium was 8.52 ± 4.01 ng/g and compression did not altered TNF‐α release (12.86 ± 7.84 ng/g). TNF‐α basal release was significantly higher in hypertrophic scar (14.74 ± 1.42 ng/g) compared to normal scar samples and TNF‐α secretion was diminished (3.52 ± 0.97 ng/g) after compression. In conclusion, in our in vitro model, mechanical compression resembling the clinical use of elastocompression was able to strongly increase apoptosis in the hypertrophic scar derma as observed during granulation tissue regression in normal wound healing. Moreover, the observed modulation of IL‐1β and TNF‐α release by mechanical loading could play a key role in hypertrophy regression induced by elastocompression. (WOUND REP REG 2003;11:331–336)
Keratinocyte outgrowth from human skin explant cultures is dependent upon p38 signaling
Authors: Stoll Stefan W. , Kansra Sanjay , Elder James T.
Published: August 2003

Organ culture of skin is known to recapitulate several early events in the process of wound healing. Here we investigate the function of p38 kinase signaling as a regulator of keratinocyte behavior in human skin organ culture. We first show that skin organ culture recapitulates the transition from migration to proliferation that is known to characterize the reepithelialization process. We next show that inhibition of p38 markedly impairs the formation of keratinocyte outgrowth in human skin explant cultures, as well as the migration of keratinocytes in an in vitro wound assay. In contrast, the marked induction of mRNA encoding the ErbB ligand heparin‐binding epidermal growth factor‐like growth factor, known to occur after skin wounding, was not blocked by inhibition of p38. As assessed by immunoblotting, phosphorylation of p38 was limited and was not increased between 0 and 7 days of organ culture. Our results show the sensitivity of reepithelialization to inhibition by p38 and suggest that p38 acts primarily during the migration phase of this process. These data also indicate that autocrine heparin‐binding epidermal growth factor expression is not regulated by p38. (WOUND REP REG 2003;11:346–353)
Bone morphogenetic protein‐2 regulates proliferation of human mesenchymal stem cells
Authors: Akino Kozo , Mineta Takao , Fukui Masashi , Fujii Tohru , Akita Sadanori
Published: August 2003

Human mesenchymal stem cells obtained from the iliac crest of a single donor were investigated for cell proliferation, cell cycle profile, gene expression, and ultrastructural changes using electron microscopy. The human mesenchymal stem cells significantly increased their cell number by day 2 after treatment with bone morphogenetic protein‐2 alone, or basic fibroblast growth factor alone or combinations of both proteins under serum‐free conditions (p < 0.01). The human mesenchymal stem cells showed marked expression of cell nuclear antigen, notably at day 1, and pituitary tumor transforming gene throughout the experiment, suggesting cell cycle progression by bone morphogenetic protein‐2 treatment. In addition, strong cellular nuclear bromodeoxyuridine incorporation was seen by immunocytochemistry. Fluorescence‐activated cell sorting also showed a similar pattern of cell cycle progression with bone morphogenetic protein‐2 treatment in serum‐free medium and 10% fetal bovine serum treatment. The bone morphogenetic protein‐2–treated human mesenchymal stem cells showed heterochromatin in the nucleus, suggesting cell differentiation, and well‐developed granular endoplasmic reticulum, indicative of protein production. Overall, the human mesenchymal stem cells successfully proliferated with appropriate cell cycle progression and the cell ultrastructural morphology suggested marked nuclear and granular endoplasmic reticulum induction by bone morphogenetic protein‐2 treatment in serum‐free medium. (WOUND REP REG 2003;11:354–360)
Influence of platelet‐derived growth factor on microcirculation during normal and impaired wound healing
Authors: Uhl Eberhard , Rösken Frank , Sirsjö Allan , Messmer Konrad
Published: August 2003

The aim of the current study was to evaluate the influence of platelet‐derived growth factor (PDGF) on skin microcirculation during normal and impaired wound healing. Secondary healing wounds were created on the ears of hairless mice and treated once with 3 µg of PDGF‐BB immediately after wound creation. Intravital fluorescence microscopy was used to quantify reepithelialization, revascularization, vessel diameters, vascular permeability, and leukocyte–endothelium interactions up to 24 days after wound creation. Microvascular perfusion was assessed by laser Doppler flowmetry. Wound healing was studied in normal (n = 15) and ischemic skin tissue (n = 15) as well as in mice (n = 17) rendered hyperglycemic by an intravenous injection of streptozotocin 7 days prior to wound creation. Treatment with PDGF accelerated reepithelialization and reduced the time for complete wound closure in ischemic skin from 14.9 ± 2.5 (control) to 12.3 ± 1.8 days (p < 0.03), and in hyperglycemic animals from 15.0 ± 2.4 (control) to 12.0 ± 3.0 days (p < 0.04). Revascularization of these wounds was also significantly enhanced after PDFG application. No other parameters were influenced by the treatment. Normal wound healing was not affected. This study confirms the positive influence of PDGF on wound healing under pathophysiological conditions. The effects in this model seem to be primarily due to the mitogenic potency of PDGF on keratinocytes and endothelial cells. A significant effect on leukocyte activation during the inflammatory process was not observed. (WOUND REP REG 2003;11:361–367)
Inhibition of prolyl 4‐hydroxylase reduces scar hypertrophy in a rabbit model of cutaneous scarring
Authors: Kim Injoong , Mogford Jon E. , Witschi Claudia , Nafissi Mehdi , Mustoe Thomas A.
Published: August 2003

Hypertrophic scars result from excessive collagen deposition at sights of healing dermal wounds and can be functionally and cosmetically problematic. Pharmacological regulation of collagen synthesis and deposition is a direct approach to the control of scar tissue formation. We tested the ability of the phenanthrolinone derivative FG‐1648 (in 0.5% Carbopol 971 PNF gel, pH 6.5), a prolyl 4‐hydroxylase inhibitor, to reduce hypertrophic scar formation in a rabbit ear hypertrophic scar model. New Zealand White rabbits were divided into two treatment groups (n = 12 wounds per group with an equal number of controls): low‐dose group: 0.5% FG‐1648; high‐dose group: 1% FG‐1648. Left ears were used for treatment and right ear for control. Four 7‐mm dermal ulcer wounds were made on each ear. The inhibitor was topically applied to the wound at the time of wounding and once daily up to postoperative day 7. Wounds were harvested at postoperative day 28 and scar hypertrophy quantified by measurement of the scar elevation index. All wounds showed complete healing. Treatment of wounds with 1% prolyl 4‐hydroxylase inhibitor decreased the scar elevation index by 26% compared to control wounds (p < 0.01). Wounds treated with 0.5% FG‐1648 inhibitor showed no difference in scar elevation compared to control wounds. These results suggest that inhibition of prolyl 4‐hydroxylase may be a suitable agent for topical treatment for the prevention of hypertrophic scar tissue. (WOUND REP REG 2003;11:368–372)
Absence of inhibition of cutaneous wound healing in mice by oral doxycycline
Authors: Hebda Patricia A. , Whaley Diana , Kim Hyung‐Gyoon , Wells Alan
Published: August 2003

Temporally controlled expression of genes in transgenic mice has advanced our understanding of many physiological processes. One of the more common modes of acutely altering gene levels involves the doxycycline‐regulated “tet‐on/tet‐off” systems. There has been concern that the administration of doxycycline in itself might compromise many aspects of wound repair. Here we report that oral ad libitum administration of doxycycline (2 mg/ml in drinking water) to mice does not alter dermal or epidermal wound healing. The healing of both full‐ and partial‐thickness skin wounds proceeded similarly regardless of doxycycline administration; in fact, collagen organization and tensile strength development appeared to accelerate in the presence of doxycycline. These data suggest that wound healing studies incorporating transgene expression can utilize tet‐on/tet‐off regulation of gene expression without interference from doxycycline. (WOUND REP REG 2003;11:373–379)
Fibrinogen inhibits fibroblast‐mediated contraction of collagen
Authors: Nien Yih‐Dar , Han Yuan‐Ping , Tawil Bill , Chan Linda S. , Tuan Tai‐Lan , Garner Warren L.
Published: August 2003

Extracellular matrix changes in composition and organization as it transitions from the provisional matrix of the fibrin/platelet plug to collagen scar in healed wounds. The manner in which individual matrix proteins affect these activities is not well established. In this article we describe the interactions of two important extracellular matrix components, fibrin and collagen, using an in vitro model of wound contraction, the fibroblast‐populated collagen lattice. We utilized different fibrinogen sources and measured tissue reorganization in floating and tensioned collagen lattices. Our results showed that both fibrin and fibrinogen decreased the contraction of fibroblast populated collagen lattices in a dose‐dependent manner. Polymerization of fibrinogen to fibrin using thrombin had no effect on this inhibition. Further, there was no effect due to changes in protein concentration, alternate components of the fibrin sealant, or the enzymatic action of thrombin. These results suggest that the initial stability of the fibrin provisional matrix is due to the fibrin, because this protein appears to inhibit contraction of the matrix. This may be important in the early phases of wound healing when clot stability is vital for hemostasis. Later, as fibrin is replaced by collagen, wound contraction can occur. (WOUND REP REG 2003;11:380–385)
Gene expression changes in an in vitro model of chondroinduction: a comparison of two methods
Authors: Yates Karen E. , Glowacki Julie
Published: August 2003

There are many useful technologies to describe patterns of gene expression that occur during tissue repair and regeneration. Results from different methods used in one experimental setting are not often compared. In this case study of chondrogenesis, we compare two methods to identify differentially expressed genes, representational difference analysis and targeted macroarray analysis, as a model for investigating genes that may be relevant to tissue repair. We sought to identify genes whose expression was altered when human dermal fibroblasts were cultured in a three‐dimensional, porous collagen sponge with the chondroinductive agent, demineralized bone. Both representational difference analysis and macroarray experiments revealed several functional families of genes as up‐regulated or down‐regulated in chondroinduced fibroblasts. An advantage of representational difference analysis is that altered expression of specific mRNA transcripts can be revealed. In this example, representational difference analysis uncovered the up‐regulation of a specific transcript of Wnt5a in fibroblasts cultured with demineralized bone. Representational difference analysis is limited, however, as there can be false negatives for genes not readily amplified by polymerase chain reaction. We conclude that small arrays containing functional classes of genes can be used to ask specific, hypothesis‐driven questions at minimal cost. It may be prudent, however, to use more than one method to survey differences in gene expression in order to validate and expand findings. (WOUND REP REG 2003;11:386–392)
Timing of high‐grade metamorphism: Early Palaeozoic U–Pb formation ages of titanite indicate long‐standing high‐T conditions at the western margin of Gondwana (Argentina, 26–29°S)
Authors: Lucassen F. , Becchio R.
Published: August 2003

Abstract Concordant U–Pb ages of c. 530–510 Ma and c. 470–420 Ma on titanite from calcsilicate, orthogneiss and amphibolite rocks constrain the age of high‐T metamorphism in the Early Palaeozoic mobile belt at the western margin of Proterozoic Gondwana (Argentina, 26–29°S). The U–Pb ages document the time of titanite formation at high‐T conditions according to the stable mineral paragenesis and occurrence of titanite in the metamorphic fabric. The presence of migmatite at all sample sites indicates temperatures were > c. 650 °C. Titanite formed at similar metamorphic conditions at different times on the regional and on the outcrop scale. The titanite crystals preserved their U–Pb isotopic signatures and chemical composition under ongoing upper amphibolite to granulite facies temperatures. Different thermal peaks or deformations are only detected by the different U–Pb ages and not by changes in the mineral paragenesis or metamorphic fabric of the samples. The range of U–Pb ages, e.g. in the Ordovician and Silurian (c. 470, 460, 440, 430, 420 Ma), is interpreted as the effect polyphase deformation with deformation‐enhanced recrystallization of titanite and/or different thermal peaks during a long‐standing, geographically fixed, high‐T regime in the mid‐crust of a continental magmatic arc. A clear correlation of the different ages with distinct tectonic events, e.g. collision of terranes, is not possible based on the present knowledge of the region.
Contrasting metamorphic evolution of metasedimentary rocks from the Çine and Selimiye nappes in the Anatolide belt, western Turkey
Authors: Régnier J. L. , Ring U. , Passchier C. W. , Gessner K. , Güngör T.
Published: August 2003

Abstract

P–T conditions, mineral isograds, the relation of the latter to foliation planes and kinematic indicators are used to elucidate the tectonic nature and evolution of a shear zone in an orogen exhumed from mid‐crustal depths in western Turkey. Furthermore, we discuss whether simple monometamorphic fabrics of rock units from different nappes result from one single orogeny or are related to different orogenies. Metasedimentary rocks from the Çine and Selimiye nappes at the southern rim of the Anatolide belt of western Turkey record different metamorphic evolutions. The Eocene Selimiye shear zone separates both nappes. Metasedimentary rocks from the Çine nappe underneath the Selimiye shear zone record maximum P–T conditions of about 7 kbar and >550 °C. Metasedimentary rocks from the overlying Selimiye nappe have maximum P–T conditions of 4 kbar and c. 525 °C near the base of the nappe. Kinematic indicators in both nappes are related to movement on the Selimiye shear zone and consistently show a top‐S shear sense. Metamorphic grade in the Selimiye nappe decreases structurally upwards as indicated by mineral isograds defining the garnet‐chlorite zone at the base, the chloritoid‐biotite zone and the biotite‐chlorite zone at the top of the nappe. The mineral isograds in the Selimiye nappe run parallel to the regional SR foliation, parallel the Selimiye shear zone and indicate that the Selimiye shear zone formed during this prograde greenschist to lower amphibolite facies metamorphic event but remained active after the peak of metamorphism. 40Ar/39Ar mica ages and the tectonometamorphic relationship with the Eocene Cyclades–Menderes thrust, which occurs above the Selimiye nappe in the study area, suggests an Eocene age of metamorphism in the Selimiye nappe.

Metasedimentary rocks of the Çine nappe 20–30 km north of the Selimiye shear zone record maximum P–T conditions of 8–11 kbar and 600–650  °C. An age of about 550 Ma is indicated for amphibolite facies metamorphism and associated top‐N shear in the orthogneiss of the Çine nappe. Our study shows that simple monophase tectonometamorphic fabrics do not always indicate a simple orogenic development of a nappe stack. Preservation in some areas and complete overprinting of those fabrics in other areas apparently occur very heterogeneously.
Lawsonite growth in the epidote blueschists from the Ile de Groix (Armorican Massif, France): a potential geobarometer
Authors: Ballevre M. , Pitra P. , Bohn M.
Published: August 2003

Abstract The garnet blueschists from the Ile de Groix (Armorican Massif, France) contain millimetre‐ to centimetre‐sized pseudomorphs consisting of an aggregate of chlorite, epidote and paragonite. The pseudomorphed phase developed at a late stage of the deformation history, because it overgrows a glaucophane–epidote–titanite foliation. Garnet growth occurred earlier than the beginning of the ductile deformation, and thus garnet is also included in the pseudomorphs. Microprobe analyses show that garnet is strongly zoned, with decreasing spessartine and increasing almandine and pyrope contents from core to rim. Grossular content is higher in garnet cores (about 35 mole%) compared to garnet rims (about 30 mole%). Blue amphibole has glaucophane compositions with a low Fe3+ content and become more magnesian when inclusions in garnet (XMg = 0.62–0.65) are compared with matrix grains (XMg = 0.67–0.70). Matrix epidote has a pistacite content of about 50 mole%. On the basis of their shape and the nature of the breakdown products, the pseudomorphs are attributed to lawsonite. A numerical model (using Thermocalc) has been developed in order to understand the reactions controlling both the growth and the breakdown of lawsonite. Lawsonite growth could have taken place through the continuous hydration reaction Chl + Ep + Pg + Qtz + Vap = Gln + Lws, followed by the fluid‐absent reaction Chl + Ep + Pg = Grt + Gln + Lws. Peak P–T conditions are estimated at about 18–20 kbar, 450 °C. This indicates that lawsonite growth took place at increasing P and T, hence can be used as a geobarometer in the buffering assemblage garnet–glaucophane–epidote. The final part of the history is recorded by lawsonite breakdown, after cessation of the ductile deformation, and recording the earliest stages of the exhumation.
Reactive flow of mixed CO2–H2O fluid and progress of calc‐silicate reactions in contact metamorphic aureoles: insights from two‐dimensional numerical modelling
Authors: Cui X. , Nabelek P. I. , Liu M.
Published: August 2003

Previous models of hydrodynamics in contact metamorphic aureoles assumed flow of aqueous fluids, whereas CO2 and other species are also common fluid components in contact metamorphic aureoles. We investigated flow of mixed CO2–H2O fluid and kinetically controlled progress of calc‐silicate reactions using a two‐dimensional, finite‐element model constrained by the geological relations in the Notch Peak aureole, Utah. Results show that CO2 strongly affects fluid‐flow patterns in contact aureoles. Infiltration of magmatic water into a homogeneous aureole containing CO2–H2O sedimentary fluid facilitates upward, thermally driven flow in the inner aureole and causes downward flow of the relatively dense CO2‐poor fluid in the outer aureole. Metamorphic CO2‐rich fluid tends to promote upward flow in the inner aureole and the progress of devolatilization reactions causes local fluid expulsion at reacting fronts. We also tracked the temporal evolution of P‐T‐XCO2conditions of calc‐silicate reactions. The progress of low‐ to medium‐grade (phlogopite‐ to diopside‐forming) reactions is mainly driven by heat as the CO2 concentration and fluid pressure and temperature increase simultaneously. In contrast, the progress of the high‐grade wollastonite‐forming reaction is mainly driven by infiltration of chemically out‐of‐equilibrium, CO2‐poor fluid during late‐stage heating and early cooling of the inner aureole and thus it is significantly enhanced when magmatic water is involved. CO2‐rich fluid dominates in the inner aureole during early heating, whereas CO2‐poor fluid prevails at or after peak temperature is reached. Low‐grade metamorphic rocks are predicted to record the presence of CO2‐rich fluid, and high‐grade rocks reflect the presence of CO2‐poor fluid, consistent with geological observations in many calc‐silicate aureoles. The distribution of mineral assemblages predicted by our model matches those observed in the Notch Peak aureole.
Prograde and retrograde history of eclogites from the Eastern Blue Ridge, North Carolina, USA
Authors: Page F. Z. , Essene E. J. , Mukasa S. B.
Published: August 2003

Abstract The prograde metamorphism of eclogites is typically obscured by chemical equilibration at peak conditions and by partial requilibration during retrograde metamorphism. Eclogites from the Eastern Blue Ridge of North Carolina retain evidence of their prograde path in the form of inclusions preserved in garnet. These eclogites, from the vicinity of Bakersville, North Carolina, USA are primarily comprised of garnet–clinopyroxene–rutile–hornblende–plagioclase–quartz. Quartz, clinopyroxene, hornblende, rutile, epidote, titanite and biotite are found as inclusions in garnet cores. Included hornblende and clinopyroxene are chemically distinct from their matrix counterparts. Thermobarometry of inclusion sets from different garnets record different conditions. Inclusions of clinozoisite, titanite, rutile and quartz (clinozoisite + titanite = grossular + rutile + quartz + H2O) yield pressures (6–10 kbar, 400–600 °C and 8–12 kbar 450–680 °C) at or below the minimum peak conditions from matrix phases (10–13 kbar at 600–800 °C). Inclusions of hornblende, biotite and quartz give higher pressures (13–16 kbar and 630–660 °C). Early matrix pyroxene is partially or fully broken down to a diopside–plagioclase symplectite, and both garnet and pyroxene are rimmed with plagioclase and hornblende. Hypersthene is found as a minor phase in some diopside + plagioclase symplectites, which suggests retrogression through the granulite facies. Two‐pyroxene thermometry of this assemblage gives a temperature of c. 750 °C. Pairing the most Mg‐rich garnet composition with the assemblage plagioclase–diopside–hypersthene–quartz gives pressures of 14–16 kbar at this temperature. The hornblende–plagioclase–garnet rim–quartz assemblage yields 9–12 kbar and 500–550 °C. The combined P–T data show a clockwise loop from the amphibolite to eclogite to granulite facies, all of which are overprinted by a texturally late amphibolite facies assemblage. This loop provides an unusually complete P–T history of an eclogite, recording events during and following subduction and continental collision in the early Palaeozoic.
Development and evolution of craniofacial patterning is mediated by eye‐dependent and ‐independent processes in the cavefish Astyanax
Authors: Yamamoto Yoshiyuki , Espinasa Luis , Stock David W. , Jeffery William R.
Published: August 2003

Summary
We studied the development and evolution of craniofacial features in the teleost fish, Astyanax mexicanus. This species has an eyed surface dwelling form (surface fish) and many different cave dwelling forms (cavefish) with various degrees of reduced eyes and pigmentation. The craniofacial features we examined are the tooth‐bearing maxillary bones, the nasal and antorbital bones, the circumorbital bones, and the opercular bones, all of which show evolutionary modifications in different cavefish populations. Manipulations of eye formation by transplantation of the embryonic lens, by lentectomy, or by removing the optic vesicle showed that eye‐dependent and ‐independent processes change both the surface fish and cavefish craniofacial skeletons. The size of the olfactory pits, which the nasal and antorbital bones define, and the size and positioning of the circumorbital bones were found to correlate with eye development. For the six suborbital bones (SO1–6), the relationship with the developing eye appears to be due to ossification initiated from foci in the suborbital canal of cranial neuromasts, whose patterning is also highly correlated with the presence or absence of an eye. By contrast, we found that the number of maxillary teeth, the number of SO3 bone elements, the positioning of SO4–6 with respect to the opercular bone, and the shape of the opercular bone are not dependent on eye formation and vary among different cavefish populations. The results suggest that evolution of the cavefish craniofacial skeleton is controlled by multiple developmental events, some a direct consequence of eye degeneration and others unrelated to loss of the eye.
Expression of muscle‐related genes and two MyoD genes during amphioxus notochord development
Authors: Urano Aki , Suzuki Miho M. , Zhang Peijun , Satoh Nori , Satoh Gouki
Published: August 2003

Summary
The notochord is one of the diagnostic features of the phylum Chordata. Despite the similarities in the early morphogenetic patterns of the notochords of various chordates, they are strikingly distinct from one another at the histological level. The amphioxus notochord is one example of an evolutionary novelty because it is made up of muscle cells. Our previous expressed sequence tag analysis, targeting messenger RNAs expressed in the adult amphioxus notochord, demonstrated that many muscle‐related genes are expressed there. To characterize amphioxus notochord cells and to gain insights into the myogenic program in the notochord, we determined the spatial and temporal expre‐ssion patterns of these muscle‐related genes during amphioxus development. We found that BbNA1 (notochord actin), Amphi‐Trop I (troponin I), Amphi‐TPmyosin (tropo‐myosin), Amphi‐MHC2 (myosin heavy chain), Amphi‐nMRLC (notochord‐specific myosin regulatory light chain), Amphi‐nTitin/MLCK (notochord‐specific titin/myosin light chain kinase), Amphi‐MLP/CRP3 (muscle LIM protein), and Amphi‐nCalponin (notochord‐specific calponin) are expres‐sed with characteristic patterns in notochord cells, including the central cells, dorsally located cells, and ventrally located cells, suggesting that each notochord cell has a unique molecular architecture that may reflect its function. In addition, we characterized two MyoD genes (Amphi‐MyoD1 and Amphi‐MyoD2) to gain insight into the genetic circuitry governing the formation of the notochord muscle. One of the MyoD genes (Amphi‐MyoD2) is expressed in the central notochord cells, and the coexistence of Amphi‐MyoD2 transcripts along with the Amphi‐MLP/CRP3 transcripts implies the participation of Amphi‐MyoD2 in the myogenic program in the notochord muscle.
Chromosomal mapping of ANTP class homeobox genes in amphioxus: piecing together ancestral genomes
Authors: Castro L. F. C. , Holland P. W. H.
Published: August 2003

Summary
Homeobox genes encode DNA‐binding proteins, many of which are implicated in the control of embryonic development. Evolutionarily, most homeobox genes fall into two related clades: the ANTP and the PRD classes. Some genes in ANTP class, notably Hox, ParaHox, and NK genes, have an intriguing arrangement into physical clusters. To investigate the evolutionary history of these gene clusters, we examined homeobox gene chromosomal locations in the cephalochordate amphioxus, Branchiostoma floridae. We deduce that 22 amphioxus ANTP class homeobox genes localize in just three chromosomes. One contains the Hox cluster plus AmphiEn, AmphiMnx, and AmphiDll. The ParaHox cluster resides in another chromosome, whereas a third chromosome contains the NK type homeobox genes, including AmphiMsx and AmphiTlx. By comparative analysis we infer that clustering of ANTP class homeobox genes evolved just once, during a series of extensive cis‐duplication events of genes early in animal evolution. A trans‐duplication event occurred later to yield the Hox and ParaHox gene clusters on different chromosomes. The results obtained have implications for understanding the origin of homeobox gene clustering, the diversification of the ANTP class of homeobox genes, and the evolution of animal genomes.
Differential regulation of msx genes in the development of the gonopodium, an intromittent organ, and of the “sword,” a sexually selected trait of swordtail fishes (Xiphophorus)
Authors: Zauner Hans , Begemann Gerrit , Marí‐Beffa Manuel , Meyer Axel
Published: August 2003

Summary
The possession of a conspicuous extension of colored ventral rays of the caudal fin in male fish of swordtails (genus Xiphophorus) is a prominent example for a trait that evolved by sexual selection. To understand the evolutionary history of this so‐called sword molecularly, it is of interest to unravel the developmental pathways responsible for extended growth of sword rays during development of swordtail males. We isolated two msx genes and showed that they are differentially regulated during sword outgrowth. During sword growth in juvenile males, as well as during testosterone‐induced sword development and fin ray regeneration in the sword after amputation, expression of msxC is markedly up‐regulated in the sword forming fin rays. In contrast, msxE/1 is not differentially expressed in ventral and dorsal male fin rays, suggesting a link between the development of male secondary sexual characters in fins and up‐regulation of msxC expression. In addition, we showed that msx gene expression patterns differ significantly between Xiphophorus and zebrafish. We also included in our study the gonopodium, a testosterone‐dependent anal fin modification that serves as a fertilization organ in males of live‐bearing fishes. Our finding that increased levels of msxC expression are associated with the testosterone‐induced outgrowth of the gonopodium might suggest either that at least parts of the signaling pathways that pattern the evolutionary older gonopodium have been coopted to evolve a sexually selected innovation such as the sword or that increased msxC expression may be inherent to the growth process of long fin rays in general.
Regulatory punctuated equilibrium and convergence in the evolution of developmental pathways in direct‐developing sea urchins
Authors: Raff Elizabeth C. , Popodi Ellen M. , Kauffman Jeffery S. , Sly Belinda J. , Turner F. Rudolf , Morris Valerie B. , Raff Rudolf A.
Published: August 2003

Summary
We made hybrid crosses between closely and distantly related sea urchin species to test two hypotheses about the evolution of gene regulatory systems in the evolution of ontogenetic pathways and larval form. The first hypothesis is that gene regulatory systems governing development evolve in a punctuational manner during periods of rapid morphological evolution but are relatively stable over long periods of slow morphological evolution. We compared hybrids between direct and indirect developers from closely and distantly related families. Hybrids between eggs of the direct developer Heliocidaris erythrogramma and sperm of the 4‐million year distant species H. tuberculata, an indirect developer, restored feeding larval structures and paternal gene expression that were lost in the evolution of the direct‐developing maternal parent. Hybrids resulting from the cross between eggs of H. erythrogramma and sperm of the 40‐million year distant indirect‐developer Pseudoboletia maculata are strikingly similar to hybrids between the congeneric hybrids. The marked similarities in ontogenetic trajectory and morphological outcome in crosses of involving either closely or distantly related indirect developing species indicates that their regulatory mechanisms interact with those of H. erythrogramma in the same way, supporting remarkable conservation of molecular control pathways among indirect developers. Second, we tested the hypothesis that convergent developmental pathways in independently evolved direct developers reflect convergence of the underlying regulatory systems. Crosses between two independently evolved direct‐developing species from two 70‐million year distant families, H. erythrogramma and Holopneustes purpurescens, produced harmoniously developing hybrid larvae that maintained the direct mode of development and did not exhibit any obvious restoration of indirect‐developing features. These results are consistent with parallel evolution of direct‐developing features in these two lineages.
Phyletic patterns of early development in gastropod molluscs
Authors: Lindberg David R. , Guralnick Robert P.
Published: August 2003

Summary Cell lineage data for 30 exemplar gastropod taxa representing all major subclades and the outgroup Polyplacophora were examined for phylogenetic signal using cladistic analysis. Most cell lineages show phyletic trends of acceleration or retardation relative to the outgroup and more basal ingroup taxa, and when coded this variation is phylo‐genetically informative. PAUP analyses of a cell lineage data set under three sets of character ordering assumptions produced similar tree topologies. The topologies of the strict consensus trees for both ordered and Dollo (near irreversibility of character transformations) character assumptions were similar, whereas the unordered character assumption recovers the least phyletic information. The cell lineage cladograms are also in agreement with the fossil record of the timing and sequence of gastropod subclade origination. A long branch lies between the Patellogastropoda+Vetigastropoda grade and the Neritopsina+Apogastropoda clade. The geological timing of this long branch is correlated with the first large‐scale terrestrially derived eutrophication of the near‐shore marine habitat, and one possible explanation for this branch may be a developmental shift associated with the evolution of feeding larvae in response to the more productive conditions in the near‐shore water column. Although character transformations are highly ordered in this data set, developmental rate characters (like all other morphological and molecular characters) are also subject to homoplasy. Finally, this study further supports the hypothesis that early development of gastropod molluscs has conserved a strong phyletic signal for about half a billion years.
Expression of anterior Hox genes during larval development of the gastropod Haliotis asinina
Authors: Hinman Veronica F. , O'Brien Elizabeth K. , Richards Gemma S. , Degnan Bernard M.
Published: August 2003

Summary
We report the spatial expression patterns of five anterior Hox genes during larval development of the gastropod mollusc Haliotis asinina, an unsegmented spiralian lophotrochozoan. Molecular alignments and phylogenetic analysis indicate that these genes are homologues of Drosophila HOM‐C genes labial, proboscipedia, zen, Deformed, and Sex combs reduced; the abalone genes are named Has‐Hox1, ‐Hox2, ‐Hox3, ‐Hox4, and ‐Hox5. Has‐Hox transcripts are first detected in the free‐swimming trochophore larval stage and restricted to the posttrochal ectoderm. Has‐Hox2, ‐Hox3, and ‐Hox4 are expressed in bilaterally symmetrical and overlapping patterns in presumptive neuroectodermal cells on the ventral side of the trochophore. Has‐Hox1 expression is restricted to a ring of cells on the dorsoposterior surface, corresponding to the outer mantle edge where new larval shell is being synthesized. There appears to be little change in the expression domains of these Has‐Hox genes in pre‐ and posttorsional veliger larvae, with expression maintained in ectodermal and neuroectodermal tissues. Has‐Hox2, ‐Hox3, ‐Hox4, and‐Hox5 appear to be expressed in a colinear manner in the ganglia and connectives in the twisted nervous system. This pattern is not evident in older larvae. Has‐Hox1 and‐Hox4 are expressed in the margin of the mantle in the posttorsional veliger, suggesting that Hox genes play a role in gastropod shell formation.
On nitric oxide signaling, metamorphosis, and the evolution of biphasic life cycles
Authors: Bishop Cory D. , Brandhorst Bruce P.
Published: August 2003

Summary
Complex life cycles are ancient and widely distributed, particularly so in the marine environment. Generally, the marine biphasic life cycle consists of pre‐reproductive stages that exist in the plankton for various periods of time before settling and transforming into a benthic reproductive stage. Pre‐reproductive stages are frequently phenotypically distinct from the reproductive stage, and the life cycle transition (metamorphosis) linking the larval and juvenile stages varies in extent of change but is usually rapid. Selection of suitable adult sites apparently involves the capacity to retain the larval state after metamorphic competence is reached. Thus two perennial and related questions arise: How are environmentally dependent rapid transitions between two differentiated functional life history stages regulated (a physiological issue) and how does biphasy arise (a developmental issue)? Two species of solitary ascidian, a sea urchin and a gastropod, share a nitric oxide (NO)‐dependent signaling pathway as a repressive regulator of metamorphosis. NO also regulates life history transitions among several simple eukaryotes. We review the unique properties of inhibitory NO signaling and propose that (a) NO is an ancient and widely used regulator of biphasic life histories, (b) the evolution of biphasy in the metazoa involved repression of juvenile development, (c) functional reasons why NO‐based signaling is well suited as an inhibitory regulator of metamorphosis after competence is reached, and (d) signaling pathways that regulate metamorphosis of extant marine animals may have participated in the evolution of larvae.
Prehospital DNR Orders: What Do Physicians in Washington Know?
Authors: Silveira Maria J. , Buell Rick A. , Deyo Richard A.
Published: September 2003

Objectives: To assess whether physicians know of Washington State's prehospital do‐not‐resuscitate (DNR) policy, 6 years after its implementation.

Design: Cross‐sectional survey.

Setting: Washington State, April 2001.

Participants: Four hundred seventy‐one practicing physicians.

Measurements: Multivariate logistic regression was used to determine relationships between physician and practice characteristics with knowledge of policies governing advance care planning.

Results: Among respondents, 60% did not know that Washington State requires an emergency medical service (EMS)‐specific DNR order authored by a physician. Seventy‐nine percent did not know that patient‐authored advance directives apply only in hospitals and medical offices.

Conclusion: The findings in this study suggest that most physicians in Washington State lack knowledge about the documentation needed for EMS personnel to forgo pre‐hospital attempts at cardiopulmonary resuscitation. Further study is needed to determine whether physician education or legislative change is necessary.
Tacrine Inhibits Topoisomerases and Dna Synthesis to Cause Mitochondrial Dna Depletion and Apoptosis in Mouse Liver
Authors: Mansouri Abdellah , Haouzi Delphine , Descatoire Véronique , Demeilliers Christine , Sutton Angela , Vadrot Nathalie , Fromenty Bernard , Feldmann Gérard , Pessayre Dominique , Berson Alain
Published: September 2003

After several weeks of treatment, levels of alanine aminotransferase (ALT) increase in 50% of patients treated with tacrine for Alzheimer's disease. We looked for progressive effects on DNA to explain delayed toxicity. We first studied the in vitro effects of tacrine on DNA replication and topoisomerase–mediated DNA relaxation. We then treated mice with doses of tacrine reproducing the human daily dose on a body area basis and studied the effects of tacrine administration for up to 28 days on hepatic DNA, mitochondrial function, and cell death. In vitro, tacrine impaired DNA polymerase γ–mediated DNA replication and also poisoned topoisomerases I and II to increase the relaxation of a supercoiled plasmid. In vivo, administration of tacrine markedly decreased incorporation of [3H]thymidine into mitochondrial DNA (mtDNA), progressively and severely depleted mtDNA, and partly unwound supercoiled mtDNA into circular mtDNA. Incorporation of [3H]thymidine into nuclear DNA (nDNA) was barely decreased, and nDNA levels were unchanged. After 12 to 28 days of treatment, administration of tacrine increased p53, Bax, mitochondrial permeability transition, cytosolic cytochrome c, and caspase–3 activity and triggered hepatocyte apoptosis and/or necrosis. In conclusion, the intercalating drug tacrine poisons topoisomerases and impairs DNA synthesis. Tacrine has been shown to accumulate within mitochondria, and it particularly targets mtDNA. After several weeks of treatment, the combination of severe mtDNA depletion and a genotoxic stress enhancing p53, Bax, and permeability transition trigger hepatocyte necrosis and/or apoptosis.
Transcriptional Regulation of the Human Transferrin Gene by Gadd153 in Hepatoma Cells
Authors: You Kyung-Ran , Liu Ming-Jie , Han Xue-Ji , Lee Zee-Won , Kim Dae-Ghon
Published: September 2003

The transcription factor CHOP/GADD153 is reportedly induced by cellular stresses such as UV light, genotoxic agents, and protein misfolding in the endoplasmic reticulum. However, the mechanism whereby induction of the GADD153 gene is linked to a downstream pathway is still unclear. Previously, we observed that a synthetic retinoid N–(4–hydroxyphenyl)retinamide (4HPR) effectively impaired cell growth and survival (induction of growth arrest and apoptosis) in human hepatoma cells, which was accompanied by over expression of GADD153. Furthermore, GADD153–transfected Hep 3B cells were growth arrested and were sensitized to drug–induced apoptosis. Thus, in this study, we used suppression subtractive hybridization (SSH) to identify GADD153 target genes that were up–regulated or down–regulated in the GADD153 transfectants. We screened 614 sequence–verified clones by Northern blotting, of which 42 genes were scored as over expressed and 17 genes as under expressed in GADD153 transfectants compared with control vector transfectants. Of those genes, 49 corresponded to known genes in public databases. Among them, we further verified that the expression of transferrin (Tf), which is a negative acute–phase protein and is essential to cell survival as a growth factor, was highly modulated by drug–induced GADD153 over expression or by in vitro transfection. GADD153 significantly antagonized the C/EBP (C/EBP–α, –β, and –δ)–mediated transcriptional activation of the Tf gene. In conclusion, Tf and other target genes identified may play a functional role in the downstream pathway of GADD153.
Potential Role of Pten Phosphatase in Ethanol–Impaired Survival Signaling in the Liver
Authors: Yeon Jong Eun , Califano Sophia , Xu Julia , Wands Jack R. , De La Monte Suzanne M.
Published: September 2003

Chronic ethanol consumption can cause sustained hepatocellular injury and inhibit the subsequent regenerative response. These effects of ethanol may be mediated by impaired hepatocyte survival mechanisms. The present study examines the effects of ethanol on survival signaling in the intact liver. Adult Long Evans rats were maintained on ethanol–containing or isocaloric control liquid diets for 8 weeks, after which the livers were harvested to measure mRNA levels, protein expression, and kinase or phosphatase activity related to survival or proapoptosis mechanisms. Chronic ethanol exposure resulted in increased hepatocellular labeling for activated caspase 3 and nuclear DNA damage as demonstrated using the TUNEL assay. These effects of ethanol were associated with reduced levels of tyrosyl phosphorylated (PY) IRS–1 and PI3 kinase, Akt kinase, and Erk MAPK activities and increased levels of phosphatase tensin homologue deleted on chromosome 10 (PTEN) mRNA, protein, and phosphatase activity in liver tissue. In vitro experiments demonstrated that ethanol increases PTEN expression and function in hepatocytes. However, analysis of signaling cascade pertinent to PTEN function revealed increased levels of nuclear p53 and Fas receptor mRNA but without corresponding increases in GSK–3 activity or activated BAD. Although fork–head transcription factor levels were increased in ethanol–exposed livers, virtually all of the fork–head protein detected by Western blot analysis was localized within the cytosolic fraction. In conclusion, chronic ethanol exposure impairs survival mechanisms in the liver because of inhibition of signaling through PI3 kinase and Akt and increased levels of PTEN. However, uncoupling of the signaling cascade downstream of PTEN that mediates apoptosis may account for the relatively modest degrees of ongoing cell loss observed in livers of chronic ethanol–fed rats.