Characterization of a new lectin involved in the protoplast regeneration of Bryopsis hypnoides

A group of coenocytic marine algae differs from higher plants, whose totipotency depends on an intact cell (or protoplast). Instead, this alga is able to aggregate its extruded protoplasm in sea water and generate new mature individuals. It is thought that lectins play a key role in the aggregation process. We purified a lectin associated with the aggregation of cell organelles in Bryopsis hypnoides. The lectin was ca. 27 kDa with a pI between pH 5 and pH 6. The absence of carbohydrate suggested that the lectin was not a glycoprotein. The hemagglutinating activity (HA) of the lectin was not dependent on the. presence of divalent cations and was inhibited by N-Acetylgalactosamine, N-Acetylglucosamine, and the glycoprotein bovine submaxillary mucin. The lectin preferentially agglutinated Gram-negative bacterium. The HA of this lectin was stable between pH 4 to pH 10. Cell organelles outside the cytoplasm were agglutinated by the addition of lectin solution (0.5 mg ml(-1)). Our results suggest that the regeneration of B. hypnoides is mediated by this lectin. We also demonstrated that the formation of cell organelle aggregates was inhibited by nigericin in natural seawater (pH 8.0). Given that nigericin dissipates proton gradients across the membrane, we hypothesize that the aggregation of cell organelles was proton-gradient dependent.

Effects of chronic experimental alcoholism on the epithelium of the uterine horn of rats (Rattus norvegicus albinus)

Sixty adult tats (Rattus norvegicus albinus) of the same age (3 months) and with a mean body weight of 228 g were divided into two experimental groups. The control group received solid diet (Purina rat chow) and tap water ad libitum. The other (alcoholic group), received the same solid diet and was allowed to drink only sugar cane brandy dissolved in 30° Gay Lussac (v/v). At the end of periods of 90, 180 and 270 days of treatment, the animals were anaesthetized with ethyl ether during estrus, weighed and sacrificed. The final mean body weights were similar in the control and alcoholic groups. The results showed intense atrophy on the lining epithelium of the endometrium of uterine horns in the alcoholic group. Important ultrastructural epithelial alterations were also observed in the female alcoholic group, such as: intense lipid droplet accumulation, increased rough endoplasmic reticulum cisternae and mitochondrial size and presence of intraepithelial neutrophils. The secretory activity of these rats was reduced. Therefore, we concluded that alcohol acts as a toxin on the epithelial layer of the rat endometrium.

Mouse extensor digitorum longus and soleus show distinctive ultrastructural changes induced by veratrine

We investigated whether veratrine (5 μl, 10 ng/kg) injected into the mouse extensor digitorum longus (EDL) (fast-twitch) and soleus (SOL) (slow-twitch) muscles provokes distinctive ultrastructural disturbances 15, 30 and 60 min later. The mitochondria in SOL were affected earlier (within 15 min) than in EDL. Swelling of the sarcoplasmic reticulum terminal cisternae was more marked in EDL than in SOL and caused distortion of sarcomeres so that fragmentation of myofilaments was more pronounced in EDL. Hypercontracted sarcomeres were seen mainly in SOL and veratrine caused infoldings of the sarcolemma only in this muscle. In both muscles, the T-tubules remained unaffected and by 60 min after veratrine most of the above alterations had reverted to normal. Pretreatment with tetrodotoxin prevented the alterations induced by veratrine. This suggests that most of the alterations resulted from the enhanced influx of Na+ into muscle fibers. These results emphasize the importance of considering the type of muscle when studying the action of myotoxic agents.

The thrombocyte aggregation process in the turtle Phrynopys hilarii (Chelonia). An ultrastructural study

This study investigates the thrombocyte aggregation process in the South American fresh water turtle (Phrynopys hilarii) using electron microscopy. Blood was taken from surgically exposed lateral neck vessels often turtles Phrynopys hilarii during the spring and summer seasons, when the mean temperature is 37°C. Blood samples were fixed with Karnovsky solution for processing by transmission electron microscopy. The turtle thrombocytes were spindle-shaped with lobulated nuclei. Prominent vesicles and canaliculi were found throughout the cytoplasm. The cytoplasm organelles showed an agranular endoplasmatic reticulum, Golgi complex near the centrioles and scattered free ribosomes. These cells are similar to bird thrombocytes but distinct from fish and frog thrombocytes. Blood clotting time was 5 min ± 30 sec measured by the Lee and White method. Structural alterations resulting from the aggregation process occurred after activation. Thrombocytes developed numerous filopodial projections, an increased number of vacuoles and changed from spindle to spherical shape. P. hilarii thrombocytes have different morphologic characteristics compared to other non-mammalian vertebrate cells. These cells can participate in the aggregation process, as observed in birds.

The ultrastructure of the guinea pig rete testis

The chambers of the rete testis (RT) of guinea pig are lined by a simple epithelium, whose cells are squamous, cubical and columnar in shape. The epithelial cells with distinct shapes were counted and the quantitative analysis of the number of these cells showed relative predominance of cubical cells. The ultrastructural observations showed predominance of membrane interdigitations among the epithelial cells. These cells present common cytoplasmic organelles. The Golgi complex polarity is typical with observation of electronlucent vesicles on the Golgi cis face closely related to rough endoplasmic reticulum (ER) lamellae, mitochondria and large number of polysomes on the Golgi trans face. These related structures present in Golgi area of RT cells suggest secretory activity which maybe occurs in the RT epithelium. Endocytotic process also occurs in the RT and this function probably concerns the uptake of substances and resorption of seminiferous fluid. Apical cilia present in RT epithelium cells are related with fluid transport and perhaps with chemoreception. Presence of spermatozoa portions enclosed into the cytoplasm of some epithelium cells has been refferred to as spermatophagy. The RT complex is mainly supported by loose connective tissue, with collagen fibres and some Leydig cells. Leydig cells are adjacent to the network channels of the septal part of the RT and apparently are able to secrete inside the RT lumen.

Prostatic stromal microenvironment and experimental diabetes

The diabetes causes alterations in various organ systems, including the male accessory sex glands. The prostate is very important in the reproductive process and it is a frequent target of malignant changes. The aim of this work was to demonstrate the histochemical and ultrastructural alterations in the prostate of diabetic animals. Two groups of animals were utilized: control and non-obese diabetic mice (NOD). Twelve days after the characterization of diabetic status the ventral prostate was collected, fixed in Karnovsky and paraformaldehyde, processed for histochemistry and TEM associated to stereology. The results showed reduction of the epithelial area and increasing of the stromal area with muscular and collagen hypertrophy in the prostatic gland. It was characterized the development of prostatic intraepithelial neoplasia, inflammatory processes and dilation of the organelles involved in the secretory process. It was concluded that diabetes besides damaging the reproductive process, affects the glandular homeostasis favoring the development of prostatic pathologies. ©2005, European Journal of Histochemistry.

Microwave-induced fast decalcification of rat bone for electron microscopic analysis: An ultrastructural and cytochemical study

Bone decalcification is a time-consuming process. It takes weeks and preservation of the tissue structure depends on the quality and velocity of the demineralization process. In the present study, a decalcification methodology was adapted using microwaving to accelerate the decalcification of rat bone for electron microscopic analysis. The ultrastructure of the bone decalcified by microwave energy was observed. Wistar rats were perfused with paraformaldehyde and maxillary segments were removed and fixed in glutaraldehyde. Half of specimens were decalcified by conventional treatment with immersion in Warshawsky solution at 4oC during 45 days, and the other half of specimens were placed into the beaker with 20 mL of the Warshawsky solution in ice bath and thereafter submitted to irradiation in a domestic microwave oven (700 maximum power) during 20 s/350 W/±37°C. In the first day, the specimens were irradiated 9 times and stored at 40°C overnight. In the second day, the specimens were irradiated 20 times changing the solution and the ice after each bath. After decalcification, some specimens were postfixed in osmium tetroxide and others in osmium tetroxide and potassium pyroantimonate. The specimens were observed under transmission electron microscopy. The results showed an increase in the decalcification rate in the specimens activated by microwaving and a reduction of total experiment time from 45 days in the conventional method to 48 hours in the microwave-aided method.

Morphologic changes and the expression of alpha-melanocyte stimulating hormone and melanocortin-1 receptor in melasma lesions: A comparative study

Melasma is a common acquired symmetrical hypermelanosis characterized by irregular light- to dark-brown macules on sun-exposed skin areas. The literature shows few studies on its physiopathogeny. However, changes in α-melanocyte stimulating hormone (α-MSH) secretion and melanocortin-1 receptor (MC1-R) expression may play a role to trigger this condition. Biopsies were taken from both melasma skin and adjacent perilesional normal skin of 44 patients. The biopsies were submitted for hematoxylin and eosin and Fontana-Masson staining and immunohistochemistry with Melan-A, α-MSH, and MC1-R, and processed for transmission electron microscopy. In some cases, they were submitted to MC1-R gene expression analysis by real-time polymerase chain reaction. Increased lymphohistiocytic infiltrate and solar elastosis, higher epidermal melanin were observed in melasma skin. Electron microscopy revealed a greater number of mature melanosomes in keratinocytes and melanocytes, and more prominent cytoplasmic organelles in melasma skin. There was no difference in melanocyte number between areas. However, melanocytes were larger and more dendritic in melasma skin. Immunohistochemistry with α-MSH and MC1-R showed significant labeling in melasmic epidermis but MC1-R messenger ribonucleic acid (RNAm) did not show significant quantitative difference between melasma and normal skin. © 2010 by Lippincott Williams & Wilkins.

Rhipicephalus sanguineus (Acari: Ixodidae) female ticks exposed to castor oil (Ricinus communis): An ultrastructural overview

Tick control has been accomplished through the use of synthetic acaricides, which has created resistant individuals, as well as contaminating the environment and nontarget organisms. Substances of plant origin, such as oils and extracts of eucalyptus and neem leaves, have been researched as an alternative to replace the synthetic acaricides. Ricinoleic acid esters from castor oil have recently been shown as a promising alternative in eliminating bacterial contamination during ethanol fermentation, by acting as an effective biocide. The same positive results have been observed when these esters are added to the food given to tick-infested rabbits. This study tested the effect of these substance on the reproductive system of Rhipicephalus sanguineus females, added to rabbit food, more specifically on oogenesis. For this, four groups were established: four control groups (CG1, CG2, CG3, and CG4) and four treatment groups (TG1, TG2, TG3, and TG4) with one rabbit in each (New Zealand White), used as hosts. After full 4 days feeding (semi-engorgement), the females were collected and had their ovaries extracted. In this study, it was observed that R. sanguineus females exposed to esters had their ovaries modified, which was demonstrated through transmission electron microscopy techniques. The addition of ricinoleic esters to the diet of tick-infested rabbits revealed how toxic such substances are for the cytoplasmic organelles of oocytes and pedicel cells. These compounds can change the morphophysiology of germ and somatic cells, consequently influencing their viability and, therefore, confirming that the ricinoleic acid esters from castor oil are a promising substance in the control of R. sanguineus. © 2012 Springer-Verlag Berlin Heidelberg.

Biomass-to-bio-products application of feruloyl esterase from Aspergillus clavatus

The structural polysaccharides contained in plant cell walls have been pointed to as a promising renewable alternative to petroleum and natural gas. Ferulic acid is a ubiquitous component of plant polysaccharides, which is found in either monomeric or dimeric forms and is covalently linked to arabinosyl residues. Ferulic acid has several commercial applications in food and pharmaceutical industries. The study herein introduces a novel feruloyl esterase from Aspergillus clavatus (AcFAE). Along with a comprehensive functional and biophysical characterization, the low-resolution structure of this enzyme was also determined by small-angle X-ray scattering. In addition, we described the production of phenolic compounds with antioxidant capacity from wheat arabinoxylan and sugarcane bagasse using AcFAE. The ability to specifically cleave ester linkages in hemicellulose is useful in several biotechnological applications, including improved accessibility to lignocellulosic enzymes for biofuel production. © 2012 Springer-Verlag Berlin Heidelberg.

New gymnospermous woods from the Permian of the Parnaíba Basin, Northeastern Brazil, Part II: Damudoxylon, Kaokoxylon and Taeniopitys

This is the second contribution on the Permian permineralized flora of the Parnaíba Basin, NE Brazil that focuses on the gymnosperm woods from the Motuca Formation. We describe five new species of gymnospermous woods Damudoxylon buritiranaense sp. nov., Damudoxylon humile sp. nov., Damudoxylon roessleri sp. nov., Kaokoxylon punctatum sp. nov. and Taeniopitys tocantinensis sp. nov., and one new form, Taeniopitys sp. 1. The woods vary widely in their anatomy, mainly in their pith features, from simple parenchymatic to solenoid piths with canals and ducts, as well as different cells, such as secretory and sclerenchymatic ones. However, they have one characteristic in common - araucarian pitting in the radial walls of the tracheids. © 2013 Elsevier B.V.

Basic ingredients for mathematical modeling of tumor growth in vitro: Cooperative effects and search for space

Based on the literature data from HT-29 cell monolayers, we develop a model for its growth, analogous to an epidemic model, mixing local and global interactions. First, we propose and solve a deterministic equation for the progress of these colonies. Thus, we add a stochastic (local) interaction and simulate the evolution of an Eden-like aggregate by using dynamical Monte Carlo methods. The growth curves of both deterministic and stochastic models are in excellent agreement with the experimental observations. The waiting times distributions, generated via our stochastic model, allowed us to analyze the role of mesoscopic events. We obtain log-normal distributions in the initial stages of the growth and Gaussians at long times. We interpret these outcomes in the light of cellular division events: in the early stages, the phenomena are dependent each other in a multiplicative geometric-based process, and they are independent at long times. We conclude that the main ingredients for a good minimalist model of tumor growth, at mesoscopic level, are intrinsic cooperative mechanisms and competitive search for space. © 2013 Elsevier Ltd.

When supply does not meet demand-ER stress and plant programmed cell death

The endoplasmic reticulum (ER) is the central organelle in the eukaryotic secretory pathway. The ER functions in protein synthesis and maturation and is crucial for proper maintenance of cellular homeostasis and adaptation to adverse environments. Acting as a cellular sentinel, the ER is exquisitely sensitive to changing environments principally via the ER quality control machinery. When perturbed, ER-stress triggers a tightly regulated and highly conserved, signal transduction pathway known as the unfolded protein response (UPR) that prevents the dangerous accumulation of unfolded/misfolded proteins. In situations where excessive UPR activity surpasses threshold levels, cells deteriorate and eventually trigger programmed cell death (PCD) as a way for the organism to cope with dysfunctional or toxic signals. The programmed cell death that results from excessive ER stress in mammalian systems contributes to several important diseases including hypoxia, neurodegeneration, and diabetes. Importantly, hallmark features and markers of cell death that are associated with ER stress in mammals are also found in plants. In particular, there is a common, conserved set of chaperones that modulate ER cell death signaling. Here we review the elements of plant cell death responses to ER stress and note that an increasing number of plant-pathogen interactions are being identified in which the host ER is targeted by plant pathogens to establish compatibility.

Endoplasmic reticulum stress and cell death mechanisms in the cell model of Huntington’s disease

This thesis clarifies important molecular pathways that are activated during the cell death observed in Huntington’s disease. Huntington’s disease is one of the most common inherited neurodegenerative diseases, which is primarily inherited in an autosomal dominant manner. HD is caused by an expansion of CAG repeats in the first exon of the IT15 gene. IT15 encodes the production of a Huntington’s disease protein huntingtin. Mutation of the IT15 gene results in a long stretch of polyQ residues close to the amino-terminal region of huntingtin. Huntington’s disease is a fatal autosomal neurodegenerative disorder. Despite the current knowledge of HD, the precise mechanism behind the selective neuronal death, and how the disease propagates, still remains an enigma. The studies mainly focused on the control of endoplasmic reticulum (ER) stress triggered by the mutant huntingtin proteins. The ER is a delicate organelle having essential roles in protein folding and calcium regulation. Even the slightest perturbations on ER homeostasis are effective enough to trigger ER stress and its adaptation pathways, called unfolded protein response (UPR). UPR is essential for cellular homeostasis and it adapts ER to the changing environment and decreases ER stress. If adaptation processes fail and stress is excessive and prolonged; irreversible cell death pathways are engaged. The results showed that inhibition of ER stress with chemical agents are able to decrease cell death and formation of toxic cell aggregates caused by mutant huntingtin proteins. The study concentrated also to the NF-κB (nuclear factor-kappaB) pathway, which is activated during ER stress. NF-κB pathway is capable to regulate the levels of important cellular antioxidants. Cellular antioxidants provide a first line of defence against excess reactive oxygen species. Excess accumulation of reactive oxygen species and subsequent activation of oxidative stress damages motley of vital cellular processes and induce cell degeneration. Data showed that mutant huntingtin proteins downregulate the expression levels of NF-κB and vital antioxidants, which was followed by increased oxidative stress and cell death. Treatment with antioxidants and inhibition of oxidative stress were able to counteract these adverse effects. In addition, thesis connects ER stress caused by mutant huntingtin to the cytoprotective autophagy. Autophagy sustains cellular balance by degrading potentially toxic cell proteins and components observed in Huntington’s disease. The results revealed that cytoprotective autophagy is active at the early points (24h) of ER stress after expression of mutant huntingtin proteins. GADD34 (growth arrest and DNA damage-inducible gene 34), which is previously connected to the regulation of translation during cell stress, was shown to control the stimulation of autophagy. However, GADD34 and autophagy were downregulated at later time points (48h) during mutant huntingtin proteins induced ER stress, and subsequently cell survival decreased. Overexpression GADD34 enhanced autophagy and decreased cell death, indicating that GADD34 plays a critical role in cell protection. The thesis reveales new interesting data about the neuronal cell death pathways seen in Huntington’s disease, and how cell degeneration is partly counteracted by various therapeutic agents. Expression of mutant huntingtin proteins is shown to alter signaling events that control ER stress, oxidative stress and autophagy. Despite that Huntington’s disease is mainly an untreatable disorder; these findings offer potential targets and neuroprotective strategies in designing novel therapies for Huntington’s disease.

Disruption of the langerin/CD207 Gene Abolishes Birbeck Granules without a Marked Loss of Langerhans Cell Function

Langerin is a C-type lectin expressed by a subset of dendritic leukocytes, the Langerhans cells (LC). Langerin is a cell surface receptor that induces the formation of an LC-specific organelle, the Birbeck granule (BG). We generated a langerin(-/-) mouse on a C57BL/6 background which did not display any macroscopic aberrant development. In the absence of langerin, LC were detected in normal numbers in the epidermis but the cells lacked BG. LC of langerin(-/-) mice did not present other phenotypic alterations compared to wild-type littermates. Functionally, the langerin(-/-) LC were able to capture antigen, to migrate towards skin draining lymph nodes, and to undergo phenotypic maturation. In addition, langerin(-/-) mice were not impaired in their capacity to process native OVA protein for I-A(b)-restricted presentation to CD4(+) T lymphocytes or for H-2K(b)-restricted cross-presentation to CD8(+) T lymphocytes. langerin(-/-) mice inoculated with mannosylated or skin-tropic microorganisms did not display an altered pathogen susceptibility. Finally, chemical mutagenesis resulted in a similar rate of skin tumor development in langerin(-/-) and wild-type mice. Overall, our data indicate that langerin and BG are dispensable for a number of LC functions. The langerin(-/-) C57BL/6 mouse should be a valuable model for further functional exploration of langerin and the role of BG.