The horseradish peroxidase technique for cell lineage studies.

The identification of cell lineage for a given cell type of a particular tissue is an important step in understanding how this process contributes to histogenesis. The importance in understanding cell lineage has relevance for both theoretical and practical reasons. For example, delineating and identifying histogenic principals is required to advance stem cell research and tissue regeneration. To document cell lineage in a given experimental preparation, a number of techniques have been developed. This typically requires the injection of a tracer compound into a founder cell. As this cell produces progeny, the tracer is passed on to the daughter cells. By detecting the tracer in the progeny or daughter cells the investigator can determine which cells originated from the cell that was originally injected with the tracer. By using such an approach it is possible to trace the developmental path from the first cells of the embryo to the specialized cells making the tissue of the adult. A number of tracer compounds have been used with good results in lineage tracing. One of these tracer compounds is horseradish peroxidase (HRP). Several variations of the technique are available depending on what species are studied or what histological requirements are made by the study. A basic technique that can be adapted to individual needs is presented. Included in this protocol on lineage tracing are the procedures for injection, fixation, and the microscope evaluation of labelled cells in the Helobdella triseralis embryo. A brief discussion of the technique will note its advantages and disadvantages. Examples of labelled cell preparations are given to illustrate the technique.     

Supplementation with L-Glutamine and L-Alanyl-L-Glutamine Changes Biochemical Parameters and Jejunum Morphophysiology in Type 1 Diabetic Wistar Rats

We evaluated the effects of the supplementation with L-glutamine and glutamine dipeptide (GDP) on biochemical and morphophysiological parameters in streptozotocin-diabetic rats. For this purpose, thirty animals were distributed into six groups treated orally (gavage) during thirty days: non diabetic rats (Control) + saline, diabetic + saline; Control + L-glutamine (248 mg/kg), Diabetic + L-glutamine (248 mg/kg), Control + GDP (400 mg/kg), Diabetic + GDP (400 mg/kg). Diabetes was induced by an intravenous injection of streptozotocin (60 mg/kg) and confirmed by fasting glucose ≥ 200 mg/dL. Physiological parameters, i.e., body mass, food intake, blood glucose, water intake, urine and faeces were evaluated during supplementation. After the period of supplementation, the animals were euthanized. The blood was collected for biochemical assays (fructosamine, transaminases, lipid profile, total protein, urea, ammonia). Moreover, the jejunum was excised and stored for morphophysiological assays (intestinal enzyme activity, intestinal wall morphology, crypt proliferative index, number of serotoninergic cells from the mucosa, and vipergic neurons from the submucosal tunica). The physiological parameters, protein metabolism and intestinal enzyme activity did not change with the supplementation with L-glutamine or GDP. In diabetic animals, transaminases and fructosamine improved with L-glutamine and GDP supplementations, while the lipid profile improved with L-glutamine. Furthermore, both forms of supplementation promoted changes in jejunal tunicas and wall morphometry of control and diabetic groups, but only L-glutamine promoted maintenance of serotoninergic cells and vipergic neurons populations. On the other hand, control animals showed changes that may indicate negative effects of L-glutamine. Thus, the supplementation with L-glutamine was more efficient for maintaining intestinal morphophysiology and the supplementation with GDP was more efficient to the organism as a whole. Thus, we can conclude that local differences in absorption and metabolism could explain the differences between the supplementation with L-glutamine or GDP.     

Succession in subtidal macrofouling assemblages of a Patagonian harbour (Argentina, SW Atlantic)

Subtidal fouling assemblages usually consist of short-lived organisms. Colonisation sequences on man-made structures may thus be greatly affected by the temporal and spatial variability of propagule supply. This study explores the influence of seasonality on succession in the macrofouling assemblage of a Patagonian harbour (Argentina, Southwest Atlantic). Replicated artificial substrata were suspended horizontally and sampled at quarterly intervals during 1?year. The influence of seasonality on 1-year-old assemblages was further analysed using additional sets of replicated panels submersed at different seasons and collected 1?year later. Upper surfaces were always dominated by ephemeral algae, while lower surfaces exhibited high coverage of filter-feeding invertebrates. Regardless of submersion length, species richness was significantly higher on lower than on upper surfaces. A significant interaction between orientation and submersion length was found for the Shannon diversity index, meaning that temporal changes in diversity depended on substratum orientation. On the lower surfaces, diversity reached a maximum after 9?months and then declined, mainly due to extensive dislodgment of two species of ascidians. On algal-dominated upper surfaces, differences in structure of annual assemblages were due to seasonal changes in the abundance of ephemeral algae. This study shows that constancy or variability of 1-year-old assemblages whose development began at different seasons depended greatly on the life history of the organisms that settled and managed to persist on both surfaces, which in turn depended on substratum orientation.     

SEASONAL VARIATION OF PHOTOSYNTHETIC PERFORMANCE AND LIGHT ATTENUATION IN ULVA CANOPIES FROM PALMONES RIVER ESTUARY1

The primary production of Ulva populations relies on their photosynthetic performance, which is dependent on the light availability under natural conditions. This study concerns the light attenuation characteristics in Ulva canopies and the seasonal photosynthetic performance of two different species (Ulva rotundata Blid., Ulva curvata (Kütz.) De Toni) blooming in the Palmones river estuary. Light within canopies differed from that reaching the surface. Light availability was reduced through the water column (at high tide) and Ulva canopies. In addition, light was spectrally filtered. As a result, the photosynthetically usable radiation (PUR) was further attenuated through Ulva canopies, increasing the photosynthetically active radiation/PUR ratio. The muddy sediment deposited on and between the Ulva thalli also drastically restricted the light availability. Thick Ulva mats are frequently found covering the intertidal mudflats, and therefore, thalli within these mats may be subjected to steep light gradients. As a consequence, individual Ulva growth rates cannot be extrapolated to estimate the primary production of Ulva canopies. Interspecific differences were observed for light-saturated photosynthetic rates (P_max) and light compensation points (L_CP), with Ulva curvata generally displaying higher values than did U. rotundata. For both species, maxima were recorded in winter for P_max, quantum yield, chlorophyll content, and absorptance, whereas minima were found in summer. Dark respiration (R_d) was not seasonally affected, and a maximum L_CP was found in summer. To extrapolate these data to field situations, the temperature dependence of photosynthesis should be considered. The Q₁₀ values were 2.44 for R_d and 1.79 for P_max, whereas the photosynthesis rate at subsaturating light levels was unaffected. The Q₁₀ values showed an enhanced respiratory rate in summer and a minimum in winter, whereas the seasonal differences on P_max were damped.     

Environmental and anthropogenic factors associated with coastal wetland differentiation in La Mancha,Veracruz, Mexico

At least 10% of the wetlands in Mexico are situated in the State of Veracruz on the central Gulf coast. The region has been intensively used for agriculture by humans for the last thousand years and today pastureland for cattle ranching is the dominant element of the landscape. We present a study of the wetland vegetation in a series of representative and internationally significant freshwater wetlands near the coastal lagoon La Mancha in Veracruz State. We obtained eleven wetland types formed by three wetland communities dominated by trees (mangrove or swamps), eight dominated by herbaceous species which we classified as marsh, five of which were native emergent marsh, one was a non-native emergent marsh and two were floating-leaved marsh. The highest diversity values were found in the Annona swamp, followed by the Sagittaria marsh and the Rhizophora–Laguncularia–Avicennia mangrove. CCA ordination showed that water level, disturbance and species number were the major factors explaining variation along axis 1, and on axis 2 conductivity, salinity, and bulk density. Wetland size was a major factor in both axes. We examined the interplay of intrinsic and extrinsic factors that give rise to the nature and diversity of wetland types that characterize the important wetland landscapes in this part of Mexico, being disturbance by human activities an important factor that favors the appearance of diverse species combinations.     

Nocturnal courtship,copulation, and egg-laying in the western grebe (<Emphasis Type="Italic">Aechmophorus occidentalis</Emphasis>) and Clark’s grebe (<Emphasis Type="Italic">Aechmophorus clarkii</Emphasis>)

Grebes (Podicipedidae) are primarily diurnal although migration occurs at night and some species forage opportunistically at night. We used motion-activated cameras to study nocturnal reproductive activities of the western grebe, Aechmophorus occidentalis, and Clark’s grebe, Aechmophorus clarkii, at Clear Lake, California, USA, during 2014–2016. During 9283 h of monitoring, 19.4% of rushing displays (n = 75) and 8.4% of copulations (n = 435), plus two weed dancing displays and four instances of egg-laying, occurred at night. Nocturnal copulation and egg-laying often occurred on empty, recently abandoned nests, but some incubating pairs of grebes also copulated at night. Nocturnal reproductive activities occurred more frequently at nests attached to submergent vegetation in open water far from shore when water levels were low than on nests attached to emergent vegetation close to shore when water levels were high, probably due to a reduced risk of predation by nocturnal mammals farther from shore. Nocturnal reproductive activities were not in response to elevated light levels at night, daylight disturbances, diurnal predation, high temperatures on hot days, or sexual interference during daylight. Instead, grebes attempted to increase mating investment and minimize parental investment by exploiting recently abandoned nests at night, and perhaps by undertaking extrapair copulations under the cover of darkness, but some pairs of grebes may simply extended routine reproductive activities into the night. Reproductive activities are probably limited at night by the need to feed during daylight and sleep at night, and by the increased risk of predation by mammals at night. Given the high costs of construction and maintenance of floating nests and the obvious benefit of quickly exploiting abandoned nests, nocturnal reproductive activities may occur more frequently in grebes and other waterbirds using floating nests than in other groups of birds whose reproductive activities occur mostly during daylight.     

Dual modulation of a potassium channel by the m1 muscarinic and beta2-adrenergic receptors

Neurotransmitter receptors alter membrane excitability and synaptic efficacy by generating intracellular signals that ultimately change the properties of ion channels. Given their critical role in controlling cell membrane potential, potassium channels are frequently the targets of modulatory signals from many different G protein-coupled receptors. However, due to the heterogeneity of potassium channel expression in vivo, it has been difficult to determine the molecular mechanisms governing the regulation of molecularly defined potassium channels. Through expression studies in Xenopus oocytes and mammalian cells, we found that the m1 muscarinic acetylcholine receptor (mAChR) potently suppresses a cloned delayed rectifier potassium channel, termed RAK, through a pathway involving phospholipase C activation and direct tyrosine phosphorylation of the RAK protein. In contrast, we found that RAK channel activity is strongly enhanced following agonist activation of beta2-adrenergic receptors; this effect requires a single PKA consensus phosphorylation site located near the amino terminus of the channel protein. These results demonstrate that a specific type of potassium channel that is widely expressed in the mammalian brain and heart is subject to both positive and negative regulation by G protein-dependent pathways.     

Surface antigen analysis of group B Neisseria meningitidis outer membrane by monoclonal antibodies: Identification of bactericidal antibodies to class 5 protein

Twenty-four monoclonal antibodies (mAbs) against group B Neisseria meningitidis surface antigens were analyzed by immunoenzymatic assays and by a bactericidal test. Two mAbs were specific to polysaccharide B and one to lipopolysaccharide. The others were directed against outer membrane proteins ranging in molecular mass from 25 to 200 kDa. The outer membrane protein epitopes recognized by the mAbs were not conformational and were located on the outer surface of the microorganism. Linear epitopes on the class 5 protein, exposed on the surface of the membrane, were able to induce bactericidal antibodies to the homologous strain. The susceptibility of Neisseria meningitidis to these antibodies was unchanged when this organism was cultivated under conditions of iron depletion. These results demonstrate that peptides derived from class 5 proteins are potentially important in synthetic peptide or in recombinant protein vaccines containing linear bactericidal epitopes.     

Pollen limitation may be a common Allee effect in marine hydrophilous plants: implications for decline and recovery in seagrasses

Oecologia - Pollen limitation may be an important factor in accelerated decline of sparse or fragmented populations. Little is known whether hydrophilous plants (pollen transport by water) suffer...