Leaf anatomy of two Anemia Sw. species (Schizaeaceae-Pteridophyte) from a rocky outcrop in Niterói, Rio de Janeiro, Brazil

The ferns Anemia tomentosa (Sav.) Sw. var. anthriscifolia (Schrad.) Mickel and Anemia villosa Humb. & Bonpl. ex Willd. are widely associated with vegetation islands on rocky outcrops in Rio de Janeiro. Both species are desiccation tolerant. The leaf anatomy of these species was examined aiming to identify morphological characteristics that would allow the establishment of these species in water-scarce environments. The plants were harvested on "Pedra de Itacoatiara" and prepared according to the usual procedures. The petiole has a uniseriate epidermis with lignified cell walls, conical stegmata, and uniseriate multicelular and glandular trichomes. In A. villosa, the stomata protrude in a respiratory line. Under the epidermis the cells have thick, lignified walls. The parenchyma has phenolic compounds and starch grains. The petiole vascular bundles are surrounded by endodermis with Casparian strips and the xylem is V-shaped (A. villosa) or arc-shaped (A. tomentosa var. anthriscifolia). The leaf blades have a uniseriate epidermis with sinuous anticlinal and convex periclinal walls, conical stegmata and chloroplasts on both surfaces. The leaf margins of A. villosa have lignified cells. The guard cells of the stomata on the abaxial surface are on the same level or are raised above ordinary epidermal cells. Multicelular uniseriate trichomes and glandular hairs were observed. The dorsiventral mesophyll has loosely packed chlorenchyma with arm-shaped and H-shaped cells. The vascular bundles are surrounded by endodermis with Casparian strips and with parenchymatic extensions towards the epidermis. Anatomical results were analyzed considering the interaction of these plants with abiotic factors.

Anatomy, ultrastructure and secretion of Hibiscus pernambucensis Arruda (Malvaceae) extrafloral nectary

This paper reports on the extrafloral nectary (EFN) of Hibiscus pernambucensis, a native shrub species occurring in mangrove and restinga along Brazil's coastline. EFNs occur as furrows with a protuberant border on the abaxial surface veins of the leaf blade. Each nectary consists of numerous secretory multicellular trichomes, epidermal cells in palisade-like arrangements and non-vascularized parenchyma tissue. Nectar secretion is prolonged, since secretion starts in very young leaves and remains up to completely expanded leaves. Reduced sugars, lipids, and proteins were histochemically detected in all the nectary cells; phenolic substances were detected in the vacuoles of the epidermal palisade cells and in some secretory trichome cells. The secretory cells that constitute the body of trichomes have large nuclei, dense cytoplasm with numerous mitochondria, dictyosomes, scattered lipid droplets and plastids with different inclusions: protein, lipid droplets or starch grains; vacuoles with different sizes have membranous material, phenolic and lipophilic substances. The palisade cells show thick periclinal walls, reduced cytoplasm with voluminous lipid drops and developed vacuoles. The nectary parenchyma cells contain abundant plasmodesmata and cytoplasm with scattered lipid droplets, mitochondria, plastids with starch grains and endoplasmic reticulum. Mucilage idioblasts are common in the inner nectary parenchyma. Protoderm and ground meristem participate in the formation of EFN. Our data indicate that all nectary regions are involved in nectar production and secretion, constituting a functional unit. Longevity of the extrafloral nectaries is likely associated with the presence of mucilage idioblasts, which increases the capacity of the nectary parenchyma to store water.

Palynotaxonomy of Vernonanthura H. Rob. (Vernonieae, Asteraceae) species from Southeast Brazil

The present work studied the pollinic characteristics of 22 species of Vernonanthura from Southeast Brazil. The pollen grains were acetolysed, measured, described and ilustrated under light, scanning and transmission electron microscopy. Based on the results, Vernonanthura was considered an euripolinic genus as pollen grains presented distinctive characteristics, mainly related to the ornamentation, the size and apertures of the pollen grains, which may serve as a further tool in the delimitation of the species.

Pollen morphology of species of Hortia (Rutaceae)

A palinological study covering all 10 species of the Neotropical genus Hortia Vand. (Rutaceae) was conducted. Pollen grains were collected from herbarium exsiccates, acetolysed and mounted in glycerine jelly on glass slides. The grains were studied under light and scanning electron microscopy, including measurements of polar and equatorial diameters, shape of the grains, number and shape of apertures, and thickness of the exine. The results demonstrated that the genus is stenopalynous, with pollen grains in monads, subprolate to prolate, 3-colporate, with very thick exine and a psilate-perforate pattern of ornamentation. Pollen grains of all species revealed a great similarity, with few variations in the pattern of ornamentation of exine, number and form of apertures and measurements. Although well-characterized palinologycally, pollen features did not furnish relevant information on the position of Hortia into an intrafamilial phylogeny.

Pollination and fruit set in pumpkin (Cucurbita pepo) by honey bees

Species of Cucurbitaceae are cultivated worldwide and are depend on bee pollination for fruit set. Field and lab experiments were conducted at Cornell University, Ithaca, NY, during 1996 and 1997 to determine "Howden" pumpkin (Cucurbita pepo L.) pollen removal and deposition by honeybees and factors relating to male flower attractiveness. Several parameters were evaluated in flowers at anthesis: (1) removal of pollen from anthers by honey bees, (2) pollen deposition on the stigma by honey bees, (3) amount of pollen on the body of honey bees, (4) fruit set after bee pollination, and (5) male flower nectary's pores and flower attractiveness. Honey bees carried between 1,050 to 3,990 pollen grains and 13,765 were removed from an anther after one visit. The amount of pollen deposited on the stigma by the honey bees varied according to the number of visits, from 53 grains with one visit, to 1,253 grains with 12 visits, and the mean number of grains in each visit varied from 53 to 230 grains. The percentage of established fruits was higher (100%) when the flowers received 12 visits of Apis mellifera, corresponding to a load 1,253 pollen grains. The attractiveness of the male flower for pollen and nectar collection was increased by the degree of opening of the access pore to the nectary in the flower.

Multivariate analysis of pollen frequency of the native species Escallonia pulverulenta (Saxifragaceae) in Chilean honeys

The aim of this work was the identification of geographic zones suitable for the production of honeys in which pollen grains of Escallonia pulverulenta (Ruiz & Pav.) Pers. (Saxifragaceae) can be detected. The analysis of botanical origin of 240 honey samples produced between La Serena and Puerto Mont (the IV and X Administrative Regions of Chile), allowed the detection of pollen grains of E. pulverulenta in 46 Chilean honeys. The geographic distribution of the honeys studied is presented together with their affinities, through factor analysis and frequency tables. The study was based on the presence of E. pulverulenta pollen. Escallonia pulverulenta pollen percentages oscillated between 0.24% and 78.5%. Seventeen of the studied samples were designated as unifloral - i.e. samples showing more than 45% pollen of a determined plant species. Two of these corresponded to E. pulverulenta (corontillo, madroño or barraco) honeys. The remaining unifloral honeys correspond to 8 samples of Lotus uliginosus Schkuhr (birdsfoot trefoil), 2 samples of Aristotelia chilensis (Molina) Stuntz (maqui) and 1 sample of Escallonia rubra (Ruiz & Pav.) Pers. (siete camisas), Eucryphia cordifolia Cav. (ulmo or muemo), Weinmannia trichosperma Cav. (tineo), Rubus ulmifolius Schott (blackberry) and Brassica rapa L. (turnip). Honeys with different percentages of E. pulverulenta pollen - statistically analyzed through correspondence analysis - could be associated and assigned to one of three geographic types, defined on the basis of this analysis. The geographical type areas defined were the Northern Mediterranean Zone (samples from the IV Region), Central Mediterranean Zone (samples from the V to the VIII regions including two samples of unifloral Escallonia pulverulenta honey), and Southern Mediterranean Zone (samples from the IX Region).

Meiotic behavior in wild and domesticated species of Passiflora

The meiotic behavior of fourteen Passiflora taxa was analyzed. The species were grouped according to the n value (6, 9 and 12) for statistical studies. Some species presented tetravalent associations or univalent chromosomes in diakinesis, bivalent formation prevailing. The qui-square test revealed significant differences in the chiasma frequency among species for n = 9 and n = 6 groups. There was predominance of interstitial chiasmata in almost all studied species. The n = 12 group was the only one whose meiotic behavior was considered similar due to the quantity of chiasmata per cell, tendency of interstitial chiasma localization. Some species presented meiotic irregularities, such as laggard and precocious chromosomes in meiosis I. In telophase II the percentages of meiotic irregularities was low. Irregularities in the spindle orientation were presented in higher percentages in the end of meiosis II, and were also responsible for post-meiotic abnormal products. The irregularities observed during meiosis can have influence on the percentage of sterile pollen grains and success of interspecific crossings in Passiflora species.

Self-, cross- and interspecific pollinations in Passiflora capsularis and P. rubra

This study aimed to characterize the reproductive system of Passiflora capsularis L. and P. rubra L. In vivo controlled pollinations, in vitro pollen germination and pollen-ovule (P:O) ratio evaluation were conducted. In self-pollination, intraspecific and interspecific pollination, P. capsularis showed means of 62.5, 68.7 and 48.4% of fertilized flowers, while in P. rubra, the averages were 67.2, 62.5 and 46.9%, respectively. For in vitro germination, 52.2% of P. capsularis pollen grains germinated while in P. rubra, the percentage was 64.4. The P:O ratio was 22.4 for P. capsularis, and 27.4 for P. rubra, which included them in the category of obligatory autogamous. Passiflora capsularis and P. rubra can reproduce both by self-pollination and cross-pollination, and crossings between the two species succeeded though the success rate was lower than 50%. The characteristics of the reproductive system of both species allow the use of greater range of options on breeding methods for production of ornamental Passiflora plants.

Pollen analysis of honey and beebread derived from Brazilian mangroves

Pollen analyses were performed on honey and beebread from hives in apiaries located in two distinct mangrove areas dominated by Laguncularia racemosa (L.) C.F. Gaernt. One apiary was located at the edge of Guanabara Bay, Rio de Janeiro State, and the other near Maranguá Bay, Bahia State, Brazil. We investigated the contribution of nectar and pollen from mangrove vegetation to Apis mellifera L. honey and beebread stocks. Intensive visitation to this plant species by honeybees and the presence of its pollen grains in honey and beebread confirmed the importance of Laguncularia racemosa as a polliniferous and nectariferous species.

Autoradiographic thyroid evaluation in short-term experimental diabetes mellitus

Previous studies have shown that in vitro thyroid peroxidase (TPO) iodide oxidation activity is decreased and thyroid T4-5'-deiodinase activity is increased 15 days after induction of experimental diabetes mellitus (DM). In the present study we used thyroid histoautoradiography, an indirect assay of in vivo TPO activity, to determine the possible parallelism between the in vitro and in vivo changes induced by experimental DM. DM was induced in male Wistar rats (about 250 g body weight) by a single ip streptozotocin injection (45 mg/kg), while control (C) animals received a single injection of the vehicle. Seven and 30 days after diabetes induction, each diabetic and control animal was given ip a tracer dose of 125I (2 µCi), 2.5 h before thyroid excision. The glands were counted, weighed, fixed in Bouin's solution, embedded in paraffin and cut. The sections were stained with HE and exposed to NTB-2 emulsion (Kodak). The autohistograms were developed and the quantitative distribution of silver grains was evaluated with a computerized image analyzer system. Thyroid radioiodine uptake was significantly decreased only after 30 days of DM (C: 0.38 ± 0.05 vs DM: 0.20 ± 0.04%/mg thyroid, P<0.05) while in vivo TPO activity was significantly decreased 7 and 30 days after DM induction (C: 5.3 and 4.5 grains/100 µm2 vs DM: 2.9 and 1.6 grains/100 µm2, respectively, P<0.05 ). These data suggest that insulin deficiency first reduces in vivo TPO activity during short-term experimental diabetes mellitus

Autoradiography reveals regional metabolic differences in the endometrium of pregnant and nonpregnant mice

The rodent endometrium undergoes remarkable modifications during pregnancy, resulting from a redifferentiation of its fibroblasts. During this modification (decidualization), the fibroblasts transform into large, polyhedral cells that establish intercellular junctions. Decidualization proceeds from the subepithelial stroma towards the deep stroma situated next to the myometrium and creates regions composed of cells in different stages of differentiation. We studied by autoradiography whether cells of these different regions have different levels of macromolecular synthesis. Radioactive amino acids or radioactive sulfate were administered to mice during estrus or on different days of pregnancy. The animals were killed 30 min after injection of the precursors and the uteri were processed for light microscope autoradiography. Silver grains were counted over cells of different regions of the endometrium and are reported as the number of silver grains per area. Higher levels of incorporation of amino acids were found in pregnant animals as compared to animals in estrus. In pregnant animals, the region of decidual cells or the region of fibroblasts transforming into decidual cells showed the highest levels of synthesis. Radioactive sulfate incorporation, on the other hand, was generally higher in nonpregnant animals. Animals without decidual cell transformation (nonpregnant and 4th day of pregnancy) showed a differential incorporation by subepithelial and deep stroma fibroblasts. This study shows that regional differences in synthetic activity exist in cells that are in different stages of transformation into decidual cells as well as in different regions of the endometrium of nonpregnant mice

Radioautographology: the proposal of a new concept

A new concept termed "radioautographology" is advocated. This term was synthesized from "radioautography" and "ology", expressing a new science derived from radioautography. The concept of radioautographology (RAGology) is that of a science whose objective is to localize radioactive substances in the biological structure of objects and to analyze and study the significance of these substances in the biological structure. On the other hand, the old term radioautography (RAG) is the technique used to demonstrate the pattern of localization of various radiolabeled compounds in specimens. The specimens used in biology and medicine are cells and tissues. They are fixed, sectioned and placed in contact with the radioautographic emulsions, which are exposed and developed to produce metallic silver grains. Such specimens are designated as radioautographs and the patterns of pictures made of silver grains are named radioautograms. The technicians who produce radioautographs are named radioautographers, while those who study RAGology are scientists and should be called radioautographologists. The science of RAGology can be divided into two parts, general RAGology and special RAGology, as most natural sciences usually can. General RAGology is the technology of RAG which consists of three fields of science, i.e., physics concerning radioactivity, histochemistry for the treatment of cells and tissues, and photochemistry dealing with the photographic emulsions. Special RAGology, on the other hand, consists of applications of general RAGology. The applications can be classified into several scientific fields, i.e., cellular and molecular biology, anatomy, histology, embryology, pathology and pharmacology. Studies carried out in our laboratory are summarized and reviewed. All the results obtained from such applications should be systematized as a new field of science in the future.

Symmetric pollen mitosis I and suppression of pollen mitosis II prevent pollen development in Brachiaria jubata (Gramineae)

Microsporogenesis and pollen development were analyzed in a tetraploid (2n = 4x = 36) accession of the forage grass Brachiaria jubata (BRA 007820) from the Embrapa Beef Cattle Brachiaria collection that showed partial male sterility. Microsporocytes and pollen grains were prepared by squashing and staining with 0.5% propionic carmine. The meiotic process was typical of polyploids, with precocious chromosome migration to the poles and laggards in both meiosis I and II, resulting in tetrads with micronuclei in some microspores. After callose dissolution, microspores were released into the anther locule and appeared to be normal. Although each microspore initiated its differentiation into a pollen grain, in 11.1% of them nucleus polarization was not observed, i.e., pollen mitosis I was symmetric and the typical hemispherical cell plate was not detected. After a central cytokinesis, two equal-sized cells showing equal chromatin condensation and the same nuclear shape and size were formed. Generative cells and vegetative cells could not be distinguished. These cells did not undergo the second pollen mitosis and after completion of pollen wall synthesis each gave rise to a sterile and uninucleate pollen grain. The frequency of abnormal pollen mitosis varied among flowers and also among inflorescences. All plants were equally affected. The absence of fertile sperm cells in a considerable amount of pollen grains in this accession of B. jubata may compromise its use in breeding and could explain, at least in part, why seed production is low when compared with the amount of flowers per raceme.

Novel Cellular Luminescence Probes for Immunological and Toxicological Assessments

Escherichia coli K-12 (pEGFPluxABCDEAmp) (E. coli-lux), constitutively emitting bioluminescence (BL), was constructed and its BL emitting properties tested in different growth and killing conditions. The BL emission directly correlated with the number of viable E. coli-lux cells, and when subjected to the antimicrobial agent, the diminishment of the BL signal was linked directly to the number of killed bacterial cells. The method provided a very convenient application, especially when compared to conventional plate counting assays. This novel real-time based method was utilized in both immunological and toxicological assessments. The parameters such as the activation phase, the lytic phase and the capacity of the killing of the serum complement system were specified not only in humans but also in other species. E. coli-lux was also successfully used to study the antimicrobial activities of insect haemolymph. The mechanisms of neutrophil activity, like that of a myeloperoxidase (MPO)-H2O2-halide system, were studied using the E. coli-lux approach. The fundamental role of MPO was challenged, since during the actual killing in described circumstances in phagolysosome the MPO system was inactivated and chlorination halted. The toxicological test system, assessing indoor air total toxicity, particularly suitable for suspected mold damages, was designed based on the E. coli-lux method. Susceptibility to the vast number of various toxins, both pure chemicals and dust samples from the buildings and extracts from molds, were investigated. The E. coli-lux application was found to possess high sensitivity and specificity attributes. Alongside the analysis system, the sampling kit for indoor dust was engineered based on the swipe stick and the container. The combination of practical specimen collector and convenient analysis system provided accurate toxic data from the dust sample within hours. Neutrophils are good indicators of the pathophysiological state of the individual, and they can be utilized as a toxicological probe due to their ability to emit chemiluminescence (CL). Neutrophils can either be used as probe cells, directly exposed to the agent studied, or they can act as indicators of the whole biological system exposed to the agent. Human neutrophils were exposed to the same toxins as tested with the E. coli-lux system and measured as luminol amplified CL emission. The influence of the toxins on the individuals was investigated by exposing rats with moniliniformin, the mycotoxin commonly present in Finnish grains. The activity of the rat neutrophils was found to decrease significantly during the 28 days of exposure.

5-Bromo-2’-deoxyuridine induces visible morphological alteration in the DNA puffs of the anterior salivary gland region of Bradysia hygida (Diptera, Sciaridae)

5-Bromo-2’-deoxyuridine (BrdUrd) has long been known to interfere with cell differentiation. We found that treatment ofBradysia hygida larvae with BrdUrd during DNA puff anlage formation in the polytene chromosomes of the salivary gland S1 region noticeably affects anlage morphology. However, it does not affect subsequent metamorphosis to the adult stage. The chromatin of the chromosomal sites that would normally form DNA puffs remains very compact and DNA puff expansion does not occur with administration of 4 to 8 mM BrdUrd. Injection of BrdUrd at different ages provoked a gradient of compaction of the DNA puff chromatin, leading to the formation of very small to almost normal puffs. By immunodetection, we show that the analogue is preferentially incorporated into the DNA puff anlages. When BrdUrd is injected in a mixture with thymidine, it is not incorporated into the DNA, and normal DNA puffs form. Therefore, incorporation of this analogue into the amplified DNA seems to be the cause of this extreme compaction. Autoradiographic experiments and silver grains counting showed that this treatment decreases the efficiency of RNA synthesis at DNA puff anlages.