Localization of carbonic anhydrase in identified glial cells of the leech central nervous system: application of histochemical and immunocytochemical methods

We localized the enzyme carbonic anhydrase (CA) in frozen sections of the leech (Hirudo medicinalis) central nervous system by two histochemical techniques and the indirect immunofluorescence technique. Hansson's cobalt precipitation method and the use of 1-dimethylamino-naphthalene-5-sulfonamide (DNSA) to build a fluorescent enzyme-substrate complex showed that glial cells are the sites of CA activity in the leech. Neuropil and connective glial cells surrounding the axons had strong CA activity, whereas packet glial cells, which surround neuron cell bodies, and neurons themselves remained unstained. Glial cells reacted markedly with FITC-coupled antibodies against CA isoenzyme II, but experiments with antibodies against CA isoenzyme I showed no reaction.     

Mid-Holocene vegetation change in the Troad (W Anatolia): man-made or natural?

New wood charcoal data from two archaeological sites in western Anatolia (Kumtepe and Troy/Çanakkale province) enabled a review of earlier reconstruction of the mid-Holocene vegetation and land use patterns in the region. Multi-proxy data from archaeology, zooarchaeology and climatology are combined to evaluate the relationship of climate-induced and man-made environmental change for a period spanning the Late Neolithic to the Early Bronze Age (ca. 5000–2450 cal b.c.). During the first settlement period (Kumtepe A: ca. 5000–4600 cal b.c.) lush vegetation with high proportions of deciduous oak and pine prevailed, enabling the intense use of natural resources by the late Neolithic population, which use might be reflected in the first few representatives of maquis vegetation. A settlement hiatus at the site between roughly 4600 and 3500 cal b.c. includes a cooling event in the Aegean and may have supported persistence and/or development of open vegetation units. However, the hiatus may have ended with a period of regeneration of the vegetation. From Kumtepe B2 (ca. 3300 cal b.c.) onward, human impact becomes clearly visible, although the main woodland taxa continue to prevail. In all, environmental and economic dynamics between 5000 and 2300 cal b.c. in the Troad can be characterised as at least two alternating developmental sequences of climate-induced vegetation change and reinforcement of woodland degradation by human activity.     

Immunological evidence that the nonhormone binding component of avian steroid receptors exists in a wide range of tissues and species

A monoclonal antibody to a fungal protein has been used to demonstrate the presence of the nonhormone binding component of molybdate-stabilized steroid receptors in a variety of vertebrate tissues. We recently identified a steroid receptor in the aquatic fungus Achlya ambisexualis where sexual morphogenesis of the male is directed by the steroid antheridiol. This receptor resembles receptors of higher organisms in exhibiting an 8S, molybdate-stabilized form. In the chick oviduct, a 90 000 molecular weight protein has previously been shown to be associated with the molybdate-stabilized complex of the progesterone receptor. We have isolated a similar protein of molecular weight about 88 000 from A. ambisexualis and have obtained a hybridomal-derived monoclonal antibody directed against it. This mouse anti-Achlya immunoglobulin G1 (IgG1) cross-reacts with the 90 000 molecular weight protein in chick oviduct cytosol and was used to detect analogous 90 000 molecular weight proteins in mammalian tissues. Tissue cytosols were incubated with antibody, and the complexes were isolated onto protein A-Sepharose. The resin-bound proteins were then analyzed by gel electrophoresis. This procedure revealed the presence of 90 000 molecular weight proteins in several mammalian tissues including rat liver, mouse liver and uterus, pig ovarian granulosa cells, human endometrium, and HeLa cells. These results demonstrate that the 90 000 molecular weight protein is not peculiar to the chick oviduct but is present in several different tissues from a variety of animals. This antibody should be a useful probe for further studies on the biological role of these proteins.     

Clutch size and the rejection of parasitic eggs: a comparative test of the maternal investment hypothesis

Evolutionary Ecology - Obligate brood parasitic birds lay their eggs in the nests of other species, reducing the host’s own reproductive output. To circumvent these fitness costs,...     

Hyaluronic acid regulates normal intestinal and colonic growth in mice

Hyaluronic acid (HA), a component of the extracellular matrix, affects gastrointestinal epithelial proliferation in injury models, but its role in normal growth is unknown. We sought to determine the effects of exogenous HA on intestinal and colonic growth by intraperitoneal injection of HA twice a week into C57BL/6 mice from 3 to 8 wk of age. Similarly, to determine the effects of endogenous HA on intestinal and colonic growth, we administered PEP-1, a peptide that blocks the binding of HA to its receptors, on the same schedule. In mice treated with exogenous HA, villus height and crypt depth in the intestine, crypt depth in the colon, and epithelial proliferation in the intestine and colon were increased. In mice treated with PEP-1, intestinal and colonic length were markedly decreased and crypt depth and villus height in the intestine, crypt depth in the colon, and epithelial proliferation in the intestine and colon were decreased. Administration of HA was associated with increased levels of EGF (intestine) and IGF-I (colon), whereas administration of PEP-1 was associated with decreased levels of IGF-I (intestine) and epiregulin (colon). Exogenous HA increases intestinal and colonic epithelial proliferation, resulting in hyperplasia. Blocking the binding of endogenous HA to its receptors results in decreased intestinal and colonic length and a mucosal picture of hypoplasia, suggesting that endogenous HA contributes to the regulation of normal intestinal and colonic growth.     

Hyaluronic acid is radioprotective in the intestine through a TLR4 and COX-2-mediated mechanism

The intestinal epithelium is sensitive to radiation injury. Damage to the intestinal epithelium is dose limiting in radiation therapy of abdominal cancers. There is a need for agents that can be given before radiation therapy to protect the intestinal epithelium. C57BL6 mice were subjected to 12 Gy of total body radiation. Some mice received intraperitoneal hyaluronic acid (HA) before radiation. Mice were killed 6 h after radiation to assess radiation-induced apoptosis in the intestine; other mice were killed at 84 h to assess crypt survival. Total body radiation (12 Gy) resulted in increased expression of HA synthases and HA in the intestine and increased plasma HA (5-fold). Intraperitoneal injection of HA (30 mg/kg) before radiation resulted in a 1.8-fold increase in intestinal crypt survival and a decrease in radiation-induced apoptosis. The radioprotective effects of HA were not seen in Toll-like receptor 4 (TLR4)- or cyclooxygenase-2 (COX-2)-deficient mice. Intraperitoneal injection of HA induced a 1.5-fold increase in intestinal COX-2 expression, a 1.5-fold increase in intestinal PGE?, and the migration of COX-2-expressing mesenchymal stem cells from the lamina propria in the villi to the lamina propria near the crypt. We conclude that 1) radiation induces increased HA expression through inducing HA synthases, 2) intraperitoneal HA given before radiation reduces radiation-induced apoptosis and increases crypt survival, and 3) these radioprotective effects are mediated through TLR4, COX-2, and the migration of COX-2-expressing mesenchymal stem cells.     

The ultrastructure of Ignicoccus: evidence for a novel outer membrane and for intracellular vesicle budding in an archaeon

A novel genus of hyperthermophilic, strictly chemolithotrophic archaea, Ignicoccus, has been described recently, with (so far) three isolates in pure culture. Cells were prepared for ultrastructural investigation by cultivation in cellulose capillaries and processing by high-pressure freezing, freeze-substitution and embedding in Epon. Cells prepared in accordance with this protocol consistently showed a novel cell envelope structure previously unknown among the Archaea: a cytoplasmic membrane; a periplasmic space with a variable width of 20 to 400 nm, containing membrane-bound vesicles; and an outer sheath, approximately 10 nm wide, resembling the outer membrane of gram-negative bacteria. This sheath contained three types of particles: numerous tightly, irregularly packed single particles, about 8 nm in diameter; pores with a diameter of 24 nm, surrounded by tiny particles, arranged in a ring with a diameter of 130 nm; and clusters of up to eight particles, each particle 12 nm in diameter. Freeze-etched cells exhibited a smooth surface, without a regular pattern, with frequent fracture planes through the outer sheath, indicating the presence of an outer membrane and the absence of an S-layer. The study illustrates the novel complex architecture of the cell envelope of Ignicoccus as well as the importance of elaborate preparation procedures for ultrastructural investigations.     

Elektronenmikroskopische Untersuchungen an den Tentakeln vonLanice conchilega (Polychaeta,Sedentaria)

Zusammenfassung 1. Die glatten Tentakel vonLanice conchilega Pallas wurden in bezug auf ihre Feinstruktur untersucht.2. Den äußeren Abschluß des Tentakels bildet eine Kutikula, die eine deutliche Zonierung aufweist. Der Kutikula liegt eine Schicht osmiophiler Granula auf.3. An die Kutikula schließt sich eine einschichtige Epidermis an. Diese enthält Sekret- und Sinneszellen.4. Die Sekretzellen treten gehäuft in der Tentakelspitze und weniger oft in dem übrigen Tentakel auf. Je nach Lage der Sekretzellen wird ein unterschiedliches Freiwerden der Sekrete beobachtet.5. Von der Tentakelspitze bis zum Mund verläuft eine Flimmerrinne. Eingehend wird der Feinbau der Kinocilien und ihrer Cilienwurzeln behandelt.6. Es werden drei Typen von Sinneszellen beschrieben, von denen wahrscheinlich zwei chemorezeptorische und eine mechanorezeptorische Funktionen wahrnehmen.7. Auf die Epidermis folgt ein Hautmuskelschlauch. Dieser besteht aus einer schwach ausgebildeten Ringmuskulatur und einer wesentlich stärkeren Längsmuskulatur.8. Den Abschluß zum Tentakelhohlraum bildet ein Coelothel mit vereinzelt auftretenden Coelothelwimpern.9. Die Coelomflüssigkeit enthält Coelomocyten, die dem Typ der Lymphocyten zugeordnet werden können.

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Electron microscope studies on the tentacles ofLanice conchilega (Polychaeta, Sedentaria)

The structure of the smooth tentacles inLanice conchilega Pallas was studied employing an electron microscope. The monolayered tentacle epithelium is covered by a zonated cuticle which bears a granular border. Specialized cells of the epithelium comprise secretory cells and three types of sensory cell (one mechanoreceptor and two chemoreceptors). The kinocilia of the ciliated grooves possess two types of rootlets, one of which connects the basal bodies of neighbouring cilia. Below the epithelium are located layers of circular and longitudinal muscles. The wall of the tentacle cavity is composed of a coelothelium which bears only few cilia. The coelomic fluid contains coelomocytes (lymphocytes).

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Mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Morphologische und feinstrukturelle Untersuchungen an dem Gonopodium vonHeterandria formosa Agassiz, 1853 (Pisces,Poeciliidae)

Zusammenfassung Das Gonopodium vonHeterandria formosa wurde licht- und elektronenmikroskopisch (Raster- und Transmission) untersucht. Die Zahl der Afterflossenstrahlen schwankt zwischen 8 und 11. Die Analis vonHeterandria formosa ist in drei Abschnitte unterteilt: a) in einen vorderen, stark reduzierten Flossenteil (Strahlen I+II), b) in einen mittleren, begat-tungsfunktionellen Teil (= Gonopodium, Strahlen III–V), c) in einen hinteren, normalen Flossenteil (Strahlen VI–VIII/IX/X/XI). Strahl III ist das Haupt-stützelement des Gonopodium. Rasterelektronenmikroskopische Aufnahmen zeigen, daß der vordere Ast des Strahls IV eine proximale Zähnung aufweist und zu einem gattungscharakteristischen Haken ausgezogen ist. Das Suspensorium vonHeterandria formosa besteht aus vier Gonapophysen, neun Interhämalstacheln und einer Anzahl von Baseosten.Den äußeren Abschluß des Gonopodium bildet ein mehrschichtiges Epithel. Die Epithelzellen sind untereinander stark verzahnt bzw. durch Desmosomen miteinander verbunden. Auf der Oberfläche der Epithelzellen befinden sich mäandrisch verlaufende Mikroleisten. In den Epithelzellen werden vereinzelt freie Nervenendigungen gefunden. Den basalen Abschluß der Epithelzellen bildet eine Basalmembran wechselnder Stärke, an die sich stellenweise eine aus antagonistisch angeordneten Kollagenfibrillen bestehende Faserschicht anschließt. Die Basalmembran umschließt den zentralen Knochenteil. In diesem verlaufen verschiedene afferente und efferente Blutgefäße. Die Endothelzellen der afferenten Kapillarën bilden schmale Lamellen, die sich zum Teil überlappen und durch gap und/oder tight junctions miteinander verbunden sind.

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Morphological and ultrastructural studies on the gonopodium ofHeterandria formosa Agassiz, 1853 (Pisces, Poeciliidae)

Summary The structure of the gonopodium ofHeterandria formosa was studied by light- and electron microscopy (scanning and transmission EM). In adults the number of the analis fin rays varies between 8 and 11. The anterior part of the fin which is extremely reduced in size, contains rays I and II, the median part modified for use in copulation is stabilized by rays III to V, whereas the posterior part revealing ordinary size, contains rays VI to VIII/IX/X/XI. Ray III is the main supporting element of the gonopodium. The anterior ramus of ray IV bears proximal serrae and is elongated, thus forming a terminal hook of genus specifity. InHeterandria formosa the suspensorium consists of 4 gonapophyses, 9 interhemal spines and a number of baseosts.The peripheral part of the dermis of the gonopodium is made up by a multilayered epithelium. Most of the epithelial cells are extremely toothed with numerous desmosomes. Microridges of the outer cells form a meandrian surface pattern. There are few free nerve endings within the epithelium. A basement membrane of varying thickness separates the epithelium from an underlying thin layer of connective tissue which surrounds the central bone tissues of the gonopodium. Blood vessels and nerve fiber bundles are embedded in the bone tissue. The endothelial cells of the afferent capillaries form thin lamellae which are overlapped in part. The contacts of the endothelial cells are made up by gap or tight junctions. The functional morphology of the gonopodium is discussed corresponding to the anatomical and ultrastructural findings.

     

Dynamic Precision Phenotyping Reveals Mechanism of Crop Tolerance to Root Herbivory

The western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte) is a major pest of maize (Zea mays) that is well adapted to most crop management strategies. Breeding for tolerance is a promising alternative to combat WCR but is currently constrained by a lack of physiological understanding and phenotyping tools. We developed dynamic precision phenotyping approaches using ¹¹C with positron emission tomography, root autoradiography, and radiometabolite flux analysis to understand maize tolerance to WCR. Our results reveal that WCR attack induces specific patterns of lateral root growth that are associated with a shift in auxin biosynthesis from indole-3-pyruvic acid to indole-3-acetonitrile. WCR attack also increases transport of newly synthesized amino acids to the roots, including the accumulation of Gln. Finally, the regrowth zones of WCR-attacked roots show an increase in Gln turnover, which strongly correlates with the induction of indole-3-acetonitrile-dependent auxin biosynthesis. In summary, our findings identify local changes in the auxin biosynthesis flux network as a promising marker for induced WCR tolerance.The western corn rootworm (WCR; Diabrotica virgifera virgifera LeConte; Supplemental Fig. S1) is a voracious pest of maize (Zea mays). Larvae hatch in the soil during late spring and immediately begin feeding on the crop’s root system. Over time, active feeding can result in substantial root damage with significant loss of water and/or nutrient uptake, thus weakening plants (Flint-Garcia et al., 2009). Plants also become highly susceptible to lodging when major damage is inflicted upon the anchoring root system. Taken together, these effects can result in significant corn yield losses and management costs totaling between $650 million to $1 billion in the U.S. annually (Flint-Garcia et al., 2009; Gray et al., 2009).History reveals the enormous resilience and adaptability of this pest and just how quickly it can evolve to overcome management strategies. For example, resistance to application of chemical pesticides, including cyclodienes (benzene hexachloride, aldrin) and organophosphates (methyl parathion), was seen over just a 10-year period of their use in Nebraska’s cornfields during the 1950s and 1990s, respectively (Ball and Weekman, 1963; Meinke et al., 1998). Alternate management practices, including rotation of corn with other crops on a seasonal basis, was generally considered the best choice for management since 1909 (Levine et al., 2002). In east/central Illinois, 95% to 98% of cropland had adopted a management strategy using only soybean as the rotation crop. Unfortunately, the enthusiastic adoption of this strategy over a broad area combined with the efficacy of the technique created a strong selection that favored a less common D. v. virgifera phenotype with reduced egg laying fidelity to cornfields. Over time, natural selection afforded a strong reproductive advantage to females laying their eggs in soybean fields. Since the late 1990s, a strain of the western corn rootworm with resistance to crop rotation can be found in parts of Illinois, Indiana, and parts of bordering states (Gray et al., 2009; Levine et al., 2002).More recently, D. v. virgifera resistance to deployed genetically modified organisms has been reported. First introduced into the market to target this pest back in 2003, genetically altered Bt-maize expressing one or more proteins from the soil bacteria Bacillus thuringiensis provided enhanced plant defenses to larval feeding. When a vulnerable insect ate the Bt-containing plant, the protein became activated in its gut, forming a toxin that paralyzed the digestive system and caused it to stop feeding. Unfortunately, resistance began to show within three generations of selection (Meihls et al., 2008).An alternative strategy to reduce the negative impact of D. v. virgifera attack without triggering counter adaptations in the pest is plant tolerance, which relies on a plant’s capacity to maintain growth and yield even in the presence of substantial damage. While D. v. virgifera-tolerant maize germplasms exhibiting slight to moderate tolerances to D. v. virgifera have been reported (Flint-Garcia et al., 2009), more effective lines are needed. Unfortunately, we know very little about the underlying mechanisms for crop tolerance. Over the years, one resounding message has been that the physiological processes affected by herbivory should be better characterized before breeding tools can be leveraged in a rational way to generate improved varieties that maintain high yields under herbivore pressure (Riedell, 1990). Rational decision making in the breeding selection process requires rigorous phenotyping; however, present phenotyping tools tell us little about the plasticity of root systems, especially when it comes to understanding mechanisms for crop tolerance to attack belowground. It was recently suggested that the timing for allocation of newly fixed carbon resources as soluble sugars between leaves, stalks, and root systems, and their coordination with mobilization of other resources including amino acids, may play significant roles in determining the ability of maize plants to survive an attack by D. v. virgifera (Orians et al., 2011; Robert et al., 2014).In this work, our systematic evaluation of the physiological, metabolic, and genetic basis for root regrowth as a tolerance trait sheds new light on the regulation of the growth hormone auxin (indole-3-acetic acid [IAA]) and its role in this process. Radioactive decay of ¹¹C (β⁺ emitter, t_1/2 = 20.4 min), dynamic whole-plant positron emission tomography, root autoradiography, and radiometabolite flux analyses allowed us to map the transport, allocation and metabolism of carbon and nitrogen resources against genetic and radiolabeled biochemical markers including [¹¹C]IAA, [¹¹C]indole, [¹¹C]indole-3-acetonitrile ([¹¹C]IAN), [¹¹C]indole-3-acetamide ([¹¹C]IAM), and l-[5-¹¹C]Gln (Supplemental Fig. S2). Taken together, these tools enabled us for the first time, to our knowledge, to rigorously map out the auxin biosynthesis flux network at regional tissue levels and in turn provide new insights on auxin regulation and its coordination with the availability of a key amino acid, l-Gln. The developed phenotyping tools can now be employed for the rapid identification and selection of D. v. virgifera-tolerant maize germplasm.