A Review of the Eyeless Mutant in the Mexican Axolotl

SYNOPSIS. As a homozygous recessive, gene e in the Mexican axolotlprevents optic vesicles from forming, thus producing an eyelessanimal. Previous experimental evidence has indicated that thegene acts by affecting the ability of anterior medullary plateectoderm in the eye field to respond to inductive mesodermalsignals. Other possible mechanisms of gene action are described.The hypothalamus is also affected and "eyeless" animals aresterile. The absence of eyes results in increased levels ofcirculating MSH and thus the animal is also highly pigmented.Eyes may be grafted into the heads of "eyeless" axolotls. Theseeyes become functional and lead to normal pigmentation. Whenpresent as a homozygous recessive, gene r acts to allow thepenetration of heterozygous e (E/e r/r). This results in abnormaleye development.     

Properties of the Cytochrome c Oxidase Activity of Cytochrome b561 from Photoanaerobically Grown Rhodopseudomonas sphaeroides

Cytochrome b₅₆₁ from Rhodopseudomonas sphaeroides had cytochromec (c2) oxidase activity and a pH optimum at 6.0 for this activity.The activity was affected by the ionic strength of the reactionmixture. The apparent K_m and maximal velocity (V_max) valuesin the absence of addea salts were 14 µM and 120 nmoloxidized per min per mg protein for horse heart cytochrome c.Reduced horse heart cytochrome c was reoxidized in first-orderkinetics by this cytochrome b₅₆₁. The specific activity was0.7 s^–1 per mg protein at 20°C at the concentrationof 30 µMM cytochrome c. Activity was inhibited by KCN and NaN₃, but not by antimycin.The addition of a low concentration of KCN to the cytochromeb₅₆₁ produced a change in the absorption spectrum, evidencethat KCN interacts with the heme moiety of cytochrome b₅₆₁.Results of this and preceeding studies show that the cytochromeoxidase (cytochrome "o") described earlier (Sasaki et al. 1970)is cytochrome b₅₆₁. (Received May 16, 1983; Accepted September 8, 1983)     

THE PHYSIOLOGICAL EFFECTS OF AQUEOUS SUSPENSIONS OF PLANT MOLLUSCICIDES ON HELISOMA DURYI (GASTROPODA: PLANORBIDAE)

The behavioural and physiological responses of Heltsoma duryito solutions of three South African medicinal, mollusricidalplants (Warburgia salutans, Gardenia thunbergia and Apodytesdimidiata subsp dimidiata) were investigated. Clinical signsof toxic-lty, oxygen consumption, heart rate and water uptakewere used to assess response to these mollus-cicides. All plantsproduced symptoms characteristic of the distress syndrome describedby Harry & Aldrich (1963). Haemolysis, however, occurredonly in response to extracts of G. thunbergia. Respiratory failurein snails exposed to the W. salutans and G. thunbergia did notappear to be the primary cause of mortality. All plants produceda significant lowering of heart rate at lethal concentrations.Water unbalance, which is a commonly suggested effect, did notoccur. The difficulty of inferring mode of action from observedeffects is emphasized. Although some responses are common tothe three plant mollusci-cides under investigation, the differencesclearly indicate the potential for alternative modes of action. (Received 14 November 1995; accepted 17 April 1996)     

Bcl-2 family members and apoptosis, taken to heart

Loss of myocardial cells via apoptosis has been observed in many cardiovascular diseases and has been shown to contribute to the initiation and progression of heart failure. The Bcl-2 family members are important regulators of the mitochondrial pathway of apoptosis. These proteins decide whether the mitochondria should initiate the cell death program and release proapoptotic factors such as cytochrome c. The Bcl-2 proteins consist of anti- and proapoptotic members and play a key role in regulating apoptosis in the myocardium. The antiapoptotic proteins have been demonstrated to protect against various cardiac pathologies, whereas the antiapoptotic proteins have been reported to contribute to heart disease. This review summarizes the current understanding of the role of Bcl-2 proteins in the heart. cardiovascular disease; cytochrome c; protein; mitochondria     

Cellular Neuronal Control of Molluscan Heart

Bivalve and gastropod molluscs undergo large changes in externalenvironmental conditions, as well as in internal state. Cardiacresponses to these changing conditions have been recorded ina variety of species. There is a general tendency for heartrate, and presumably cardiac output, to increase in responseto situations that would increase the load on respiratory andexcretory systems. Changes in molluscan heart function in manycases appear not to be mediated directly by cardiac nerves,but rather by such indirect mechanisms as changes in blood constituentsor mechanical, hemodynamic effects on heart muscle. Three typesof cardiac response in Aplysia have been shown to be mediated,at least in part, by the heart regulator nerves. The neural circuits that regulate heart rate in Aplysia andin Helix have been partially described in cellular detail. InMercenaria, Aplysia and other molluscan species there is evidencethat cyclic adenosine monophosphate has a role in mediatingthe excitatory effects of serotonin on heart muscle. There appearsto be, in fact, a general tendency in the Aplysia nervous systemfor neurons that exert tonic, modulatory effects within neuralnetworks that control a variety of behaviors to use serotoninfor a transmitter. In each case there is evidence to suggestthat changes in cyclic adenosine monophosphate levels may mediatesome of the modulatory effects of serotonin.     

Energetic constraints and foraging efficiency

Previous research considers foraging options that differ interms of their gross rate of gain b and rate of energy expenditurec. This research argues that maximizing efficiency b/c willmaximize net energetic gain when there is an upper limit onthe amount of energy that can be assimilated. This analysisdoes not include the expenditure during the time for which theanimal is unable to forage because of this constraint. Whenthis expenditure is included, maximizing efficiency is no longeroptimal. Instead the best feeding option is the one with thehighest value of b/(c – c₁), where c, is the metabolicrate when the animal is not foraging.     

Effect of microgravity on the expression of mitochondrial enzymes in rat cardiac and skeletal muscles

Connor, Michael K., and David A. Hood. Effect ofmicrogravity on the expression of mitochondrial enzymes in rat cardiac and skeletal muscles. J. Appl.Physiol. 84(2): 593-598, 1998.The purpose ofthis study was to examine the expression of nuclear and mitochondrialgenes in cardiac and skeletal muscle (triceps brachii) in response toshort-duration microgravity exposure. Six adult male rats were exposedto microgravity for 6 days and were compared with six ground-basedcontrol animals. We observed a significant 32% increase in heartmalate dehydrogenase (MDH) enzyme activity, which was accompanied by a62% elevation in heart MDH mRNA levels after microgravity exposure.Despite modest elevations in the mRNAs encoding subunits III, IV, andVIc as well as a 2.2-fold higher subunit IV protein content afterexposure to microgravity, heart cytochromec oxidase (CytOx) enzyme activityremained unchanged. In skeletal muscle, MDH expression was unaffectedby microgravity, but CytOx activity was significantly reduced 41% bymicrogravity, whereas subunit III, IV, and VIc mRNA levels and subunitIV protein levels were unaltered. Thus tissue-specific (i.e., heart vs.skeletal muscle) differences exist in the regulation of nuclear-encoded mitochondrial proteins in response to microgravity. In addition, theexpression of nuclear-encoded proteins such as CytOx subunit IV andexpression of MDH are differentially regulated within a tissue. Ourdata also illustrate that the heart undergoes previously unidentifiedmitochondrial adaptations in response to short-term microgravityconditions more dramatic than those evident in skeletal muscle. Furtherstudies evaluating the functional consequences of these adaptations inthe heart, as well as those designed to measure protein turnover, arewarranted in response to microgravity.

     

Morphological, Biochemical and Immunohistochemical Studies on Heart Development in Cardiac Mutant Axolotls, Ambystoma mexicanum

SYNOPSIS. A naturally-occurring genetic mutation, designatedc for "cardiac lethal" in axolotls, Ambystoma mexicanum, isproving to be a useful model for studying myofibrillogenesisin differentiating heart cells. In this paper I describe morphological,biochemical and immunofluorescence studies which compare thecontractile proteins in normal and mutant hearts. In addition,morphological studies on anterior endoderm, an important heartinductor tissue in salamanders, are reviewed. Detailed electronmicroscopic studies show that normal heart myocytes containnumerous well-organized myofibrils. Although mutant heart cellscontain a few myosin and actin filaments, there are no organizedmyofibrils. Instead, amorphous proteinaceous collections areprominent in the peripheral cytoplasm of the cell where myofibrilswould be expected to first form. SDS-polyacrylamide gel electrophoresisshows that actin is present in almost normal amounts in mutanthearts, myosin heavy chain is reduced and tropomyosin is virtuallyabsent. Immunofluorescence studies reveal that myosin, -actininand tropomyosin are located prominently in theorganized myofibrilsof normal heart cells. In mutant hearts myosin is localizedalmost exclusively in the amorphous collections at the cellperipheries, -actinin also is distributed mainly in the peripheralcell cytoplasm. There is almost no staining for tropomyosin.Heavy meromyosin (HMM) binding experiments demonstrate thatthe actin in mutant heart cells is contained within the amorphouscollections in a non-filamentous state and the addition of HMMcauses its polymerization into filaments. In view of these findings,we undertook studies to determine whether there might be a causalrelationship between theabsence of tropomyosin in mutants andthe failure of actin to form into filaments. Our results indeedshow that addition of tropomyosin to glycerinated mutant heartsor homogenates of mutant hearts causes the amorphous actin toform into filaments. Thus, this single gene mutation resultsin mutant heart cells having reduced, but significant, amountsof myosin and actin, even though non-filamentous, and substantialamounts of -actinin. There is almost no tropomyosin. It is impliedthat the drastic reduction of tropomyosin in mutant cells issomehow related to the failure of normal myofilament formation,which in turn would seem to be an essential step in the normalorganization of myofibrils.     

Genie Control of Axolotl Metamorphosis

SYNOPSIS. Neoteny in the Mexican axolotl, Ambystoma mexicanum,is caused by homozygosity for a single recessive gene. The dominantallele causing physical metamorphosis is found in the closelyrelated species, Ambystoma tigrinum, with which it can hybridize.Despite the failure of axolotls to undergo physical metamorphosis,they do undergo a cryptic metamorphosis. A larval-to-adult hemoglobinform change, serum protein changes and other physiological eventsusually associated with amphibian metamorphosis occur duringearly larval life at ages comparable to the age at which Ambystomatigrinum undergoes both the cryptic and external metamorphicevents. Axolotl cryptic metamorphosis can be induced precociouslyby immersion of the larvae in low concentrations of thyroxine;physical metamorphosis can be induced with higher thyroxineconcentrations. The site of action of the gene responsible foraxolotl neoteny has not been identified. A change in the sensitivityof external metamorphic processes to thyroxine, or reduced hormonalstimulation by the pituitary or hypothalamus may be responsible.A comparison of these functions in Ambystoma tigrinum and theaxolotl may identify the lesion.     

Development of Cardiac Function in Crustaceans: Patterns and Processes

Patterns and mechanisms involved in the onset and developmentof cardiac function in a number of crustacean groups are criticallyreviewed. Irrespective of phylogeny, heart design and ecology,the onset of heart beat seems inextricably linked to the ontogenyof the thoracic segments where the heart is located. Initiallythe beat is erratic but soon becomes regular and the rate increasesas development proceeds. However, still early in developmentthe relationship between heart rate and body size shifts froma positive to a negative one. Nevertheless cardiac output continuesto increase with increasing development, via increasing strokevolume. Some species in more ‘primitive’ groupsdevelop and retain a myogenic heart beat. Others, with globularand tubular hearts, exhibit a shift from myogenicity to neurogenicityaround the time the body size vs. heart rate relationship becomesnegative. Very early cardiac function seems generally insensitiveto external factors, such as temperature, oxygen and pollutants.Sensitivity to environmental factors increases with development,perhaps over the same timescale as the cardiac regulatory mechanismsappear.     

Cruzipain,a major Trypanosoma cruzi antigen,promotes arginase-2 expression and survival of neonatal mouse cardiomyocytes

An intense myocarditis is frequently found in the acute phase of Trypanosoma cruzi infection. Despite the cardiac damage, infected individuals may remain asymptomatic for decades. Thus T. cruzi may directly prevent cardiomyocyte death to keep heart destruction in check. Recently, it has been shown that Schwann cell invasion by T. cruzi, their prime target in the peripheral nervous system, suppressed host cell apoptosis caused by growth factor deprivation. Likewise, the trans-sialidase of T. cruzi reproduced this antiapoptotic activity of the parasite. In this study, we have investigated the effect of cruzipain, another important T. cruzi antigen, on survival and cell death of neonatal BALB/c mouse cardiomyocyte cultures. We have found that cruzipain, as well as T. cruzi infection, promoted survival of cardiomyocytes cultured under serum deprivation. The antiapoptotic effect was mediated by Bcl-2 expression but not by Bcl-xL expression. Because arginase activity is involved in cell differentiation and wound healing in most cell types and it favors parasite growth within the cell, we have further investigated the effect of cruzipain on the regulation of L-arginine metabolic pathways. Our results have revealed that cruzipain enhanced arginase activity and the expression of arginase-2 isoform but failed to induce nitric oxide synthase activity. In addition, the inhibition of arginase activity by N^G-hydroxy-L-arginine, abrogated the antiapoptotic action of cruzipain. The results demonstrate that cruzipain may act as a survival factor for cardiomyocytes because it rescued them from apoptosis and stimulated arginase-2. apoptosis; Bcl-2; Bcl-xL; nitric oxide synthase; nitric oxide     

The RHG Gene Is Involved in Root and Hypocotyl Gravitropism in Arabidopsis thaliana

In higher plants, shoots show negative gravitropism and rootsshow positive gravitropism. To elucidate the molecular mechanismsof root and hypocotyl gravitropism, we segregated the secondmutation from the original phyB-1 mutant line which impairedboth root and hypocotyl gravitropism and characterized thisnovel mutation named rhg (for root and hyzypocotyl gravitropism).The rhg is a single recessive nuclear mutation and it is mappedon the lower part of the chromosome 1. Analyses on the gravitropicresponses of the rhg mutant indicate that root and hypocotylgravitropism are severely impaired but inflorescence stem gravitropismis not affected by the rhg mutation. In the rhg mutant seedlings,amyloplasts (statoliths for gravity-perception) were presentin the presumptive statocytes of roots and hypocotyls. Phototropismby roots and hypocotyls was not impaired in the rhg mutant.These results suggest that the RHG gene product probably actson the gravity-perception and/or the gravity-signal transductionin root and hypocotyl gravitropism. This is the first reportabout the genetic locus specifically involved in both root andhypocotyl gravitropism but not inflorescence stem gravitropism,supporting our hypothesis that the mechanisms of gravitropismare genetically different between hypocotyls and inflorescencestems. (Received March 11, 1997; Accepted April 17, 1997)     

Effect of pH, ionic charge, and osmolality on cytochrome c-mediated caspase-3 activity

Cytochromec-mediated activation of caspase-3 is the final commonpathway for most signals that induce apoptosis. Before release of cytochrome c from mitochondria, K⁺ andCl efflux and intracellular acidification must occur. Wehave utilized an in vitro assay to examine the role of pH, cations,anions, and uncharged molecules on the process of cytochromec-mediated activation of procaspase-3. In this cell-freesystem, a pH above 7.4 severely suppressed the activation ofprocaspase-3 but not the activity of caspase-3. KCl, NaCl, and othersalts all inhibited caspase activation, but uncharged molecules didnot. Comparison of the inhibitory capacity of various salts suggeststhat the crucial element in causing suppression is the cation. Theinhibition of alkaline pH could be overcome by increasingconcentrations of cytochrome c, whereas the inhibition ofionic charge could not, suggesting that pH and salts affect theactivation of caspase-3 by different mechanisms.

     

Pigment Bodies in Fruits of Crimson and High Pigment Lines of Tomatoes

Pigment bodies in fruits of crimson (og^c), high pigment (hp),and crimson-high pigment (og^c hp) lines of tomatoes were observedby electron and light microscopy and compared with those ofnormal red lines and a yellow cultivar. During chloroplast-chromoplasttransformation, two main structurally distinct bodies are produced,their total and relative amounts apparently accounting for theentire range of colours (from very deep red to yellow) characterizingthe mature fruits of these different colour lines. The longnarrow crystalloids, believed to be lycopene, form in associationwith an extended thylakoid system; in senescing (over-ripe)fruit many of these are reduced to shorter irregular forms.The rounded globules are believed to be beta-carotene dissolvedin lipid material derived from membrane lysis. Analytical resultscorroborate microscopic observations that the effect of theog^c gene, as compared with the r⁺ gene for normal red colour,is to increase the lycopene content and lower the beta-carotenecontent. The effect of the hp gene is to increase the levelsof both pigments. The results support the view that the genescontrol the development of fruit pigments which affect chromoplastultrastructure. Lycopersicon esculentum Mill, tomato, fruit, pigment bodies, beta-carotene, lycopene     

A novel protein involved in heart development in <Emphasis Type="Italic">Ambystoma mexicanum</Emphasis> is localized in endoplasmic reticulum

The discovery of the naturally occurring cardiac non-function (c) animal strain in Ambystoma mexicanum (axolotl) provides a valuable animal model to study cardiomyocyte differentiation. In homozygous mutant animals (c/c), rhythmic contractions of the embryonic heart are absent due to a lack of organized myofibrils. We have previously cloned a partial sequence of a peptide cDNA (N1) from an anterior-endoderm-conditioned-medium RNA library that had been shown to be able to rescue the mutant phenotype. In the current studies we have fully cloned the N1 full length cDNA sequence from the library. N1 protein has been detected in both adult heart and skeletal muscle but not in any other adult tissues. GFP-tagged expression of the N1 protein has revealed localization of the N1 protein in the endoplasmic reticulum (ER). Results from in situ hybridization experiments have confirmed the dramatic decrease of expression of N1 mRNA in mutant (c/c) embryos indicating that the N1 gene is involved in heart development.     

Hypoxia induces apoptosis via two independent pathways in Jurkat cells: differential regulation by glucose

Glucose uptakeand metabolism inhibit hypoxia-induced apoptosis in a varietyof cell types, but the underlying molecular mechanisms remain poorlyunderstood. In the present study, we explore hypoxia-mediated celldeath pathways in Jurkat cells in the presence and absence ofextracellular glucose. In the absence of extracellular glucose, hypoxiacaused cytochrome c release, caspase 3 andpoly(ADP-ribose)polymerase cleavage, and DNA fragmentation; thisapoptotic response was blocked by the caspase 9 inhibitorz-LEHD-FMK. The presence of extracellular glucose during hypoxiaprevented cytochrome c release and activation of caspase 9 but did not prevent apoptosis in Jurkat cells. In theseconditions, overexpression of the caspase 8 inhibitor v-FLIP preventedhypoxia-mediated cell death. Thus hypoxia can stimulate twoapoptotic pathways in Jurkat cells, one dependent on cytochrome c release from mitochondria that is prevented by glucoseuptake and metabolism, and the other independent of cytochromec release and resulting from activation of the deathreceptor pathway, which is accelerated by glucose uptake and metabolism.

     

Focused Microarray Analysis of Peripheral Mononuclear Blood Cells from Churg-Strauss Syndrome Patients

DNA diagnostics are useful but are hampered by difficult ethicalissues. Moreover, it cannot provide enough information on theenvironmental factors that are important for pathogenesis ofcertain diseases. However, this is not a problem for RNA diagnostics,which evaluate the expression of the gene in question. We herereport a novel RNA diagnostics tool that can be employed withperipheral blood mononuclear cells (PBMCs). To establish thistool, we identified 290 genes that are highly expressed in normalPBMCs but not in TIG-1, a normal human fibroblast cell. Thesegenes were entitled PREP after predominantly expressed in PBMCand included 50 uncharacterized genes. We then conducted PREPgene-focused microarray analysis on PBMCs from seven cases ofChurg–Strauss syndrome (CSS), which is a small-vesselnecrotizing vasculitis. We found that PREP135 (coactosin-likeprotein), PREP77 (prosaposin), PREP191 (cathepsin D), PREP234(c-fgr), and PREP136 (lysozyme) were very highly up-regulatedin all seven CSS patients. Another 28 genes were also up-regulated,albeit more moderately, and three were down-regulated in allCSS patients. The nature of these up- and down-regulated genessuggest that the immune systems of the patients are activatedin response to invading microorganisms. These observations indicatethat focused microarray analysis of PBMCs may be a practical,useful, and low-cost bedside diagnostics tool.