Neutron spectra due (13)N production in a PET cyclotron.

Monte Carlo and experimental methods have been used to characterize the neutron radiation field around PET (Positron Emission Tomography) cyclotrons. In this work, the Monte Carlo code MCNPX was used to estimate the neutron spectra, the neutron fluence rates and the ambient dose equivalent (H*(10)) in seven locations around a PET cyclotron during (13)N production. In order to validate these calculations, H*(10) was measured in three sites and were compared with the calculated doses. All the spectra have two peaks, one above 0.1MeV due to the evaporation neutrons and another in the thermal region due to the room-return effects. Despite the relatively large difference between the measured and calculated H*(10) for one point, the agreement was considered good, compared with that obtained for (18)F production in a previous work.

An alkaline thermostable recombinant Humicola grisea var. thermoidea cellobiohydrolase presents bifunctional (endo/exoglucanase) activity on cellulosic substrates.

Humicola grisea var. thermoidea is a deuteromycete which secretes a large spectrum of hydrolytic enzymes when grown on lignocellulosic residues. This study focused on the heterologous expression and recombinant enzyme analysis of the major secreted cellulase when the fungus is grown on sugarcane bagasse as the sole carbon source. Cellobiohydrolase 1.2 (CBH 1.2) cDNA was cloned in Pichia pastoris under control of the AOX1 promoter. Recombinant protein (rCBH1.2) was efficiently produced and secreted as a functional enzyme, presenting a molecular mass of 47 kDa. Maximum enzyme production was achieved at 96 h, in culture medium supplemented with 1.34 % urea and 1 % yeast extract and upon induction with 1 % methanol. Recombinant enzyme exhibited optimum activity at 60 °C and pH 8, and presented a remarkable thermostability, particularly at alkaline pH. Activity was evaluated on different cellulosic substrates (carboxymethyl cellulose, filter paper, microcrystalline cellulose and 4-para-nitrophenyl ß-D-glucopyranoside). Interestingly, rCBH1.2 presented both exoglucanase and endoglucanase activities and mechanical agitation increased substrate hydrolysis. Results indicate that rCBH1.2 is a potential biocatalyst for applications in the textile industry or detergent formulation.

Cloning, characterization and heterologous expression of the first Penicillium echinulatum cellulase gene.

AIMS: Penicillium echinulatum is effective for bioconversion processes. However, nothing is known about the molecular biology of its cellulolytic system. We describe for the first time the isolation, cloning and expression of a P. echinulatum cellulase cDNA (Pe-egl1) encoding a putative endoglucanase. METHODS AND RESULTS: Pe-egl1 cDNA was identified from random sequencing of a P. echinulatum cDNA library. The deduced EGL1 protein possibly belongs to the glycosyl hydrolase family 5A, with 387 amino acid residues and strong similarity with other fungal endoglucanases. The cDNA was heterologously expressed in Pichia pastoris. The recombinant EGL1 secreted by a Pic. pastoris recombinant strain revealed the characteristics of particular interest: an optimal activity over a broad pH range (5.0-9.0), and an optimal temperature of 60 degrees C. The recombinant EGL1 also showed high thermostability (84% of residual activity after 1 h of pre-incubation at 70 degrees C). Calcium exerted a strong stimulatory effect over EGL1 activity. CONCLUSIONS: Altogether, these results point to the potential application of this P. echinulatum endoglucanase in cellulose processing industries, particularly the textile one because of its biochemical properties. SIGNIFICANCE AND IMPACT OF THE STUDY: The characterization and heterologous expression of the first P. echinulatun cDNA inaugurates the exploitation of this potential industrial micro-organism.

Structural features of forming and developing blood capillaries of the enamel organ of rat molar tooth germs observed by light and electron microscopy.

The process of vascularization of the enamel organ, a unique epithelial structure, occurs when the tooth germ is fully developed, i.e., at the onset of dentinogenesis. Although the three-dimensional organization of the capillaries has been previously investigated, the structural features underlying the formation of the new capillaries remains poorly understood. Thus, in the hope of better understanding the mechanism of formation of the stellate reticulum capillaries, upper first molar tooth germs of newborn and 3-day-old rats were fixed in glutaraldehyde-formaldehyde and processed for light and electron microscopy. Our results showed that blood capillaries are initially in close proximity to the outer enamel epithelium. Between and intercalated with the capillaries are round/ovoid clusters of cells, some of which are vacuolated, closely apposed to the outer enamel epithelium. The outer enamel epithelium is not a continuous layer, but exhibits gaps between the cells. This suggests that the capillaries penetrate the enamel organ through these gaps, since no invagination of the epithelium was observed. The presence of a cluster of cells containing vacuoles suggests that vasculogenesis is taking place. Images showing loss of the basal lamina, proliferation of endothelial cells, presence of filopodia and lateral sprouting suggests that angiogenesis is also occurring. Thus, neoformation of capillaries of the molar enamel organ of rat seems to occur simultaneously by mechanisms of vasculogenesis and angiogenesis.

Expression and processing of a major xylanase (XYN2) from the thermophilic fungus Humicola grisea var. thermoidea in Trichoderma reesei.

AIMS: To express a gene encoding a heterologous fungal xylanase in Trichoderma reesei. METHODS AND RESULTS: Humicola grisea xylanase 2 (xyn2) cDNA was expressed in Trichoderma reesei under the main cellobiohydrolase I (cbh1) promoter (i) as a fusion to the cellobiohydrolase I (CBHI) secretion signal and (ii) the mature CBHI core-linker. The recombinant xylanase (HXYN2) was secreted into the cultivation medium and processed in a similar fashion to the endogenous T. reesei xylanases, resulting in an active enzyme. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: HXYN2 was successfully processed in T. reesei. Composition of the culture medium affected the HXYN2 yields, favouring Avicel-lactose as a carbon source. Best yields (about 0.5 g l(-1)) in shake flask cultivations were obtained from a transformant where xyn2 was fused directly to the CBHI secretion signal.

Mineralized dermal layer of the Brazilian tree-frog Corythomantis greeningi.

Some species of anuran amphibians possess a calcified dermal layer (the Eberth-Kastschenko layer) located between the "stratum spongiosum" and the "stratum compactum." This layer consists of calcium phosphate deposits, proteoglycans, and glycosaminoglycans. Although regarded as a protective layer against desiccation, a calcium reservoir, or possibly a remnant of a dermal skeleton present in anuran ancestors, very little is known about its origin, structure, and function. Thus, we studied the structure and composition of the mineralized dermal layer of Corythomantis greeningi, a peculiar hylid from the Brazilian semiarid region (caatinga), using conventional and cryosubstitution methods combined with transmission, scanning, and analytical electron microscopy. Results show that the dermal layer consists of dense, closely juxtaposed, globular structures. Although the electron opacity of the globules was variable, depending on the type of preparation, crystal-like inclusions were present in all of them, as confirmed by dark field microscopy. Electron probe X-ray microanalysis showed calcium, phosphorus, and oxygen, and electron diffraction revealed a crystalline structure comparable to that of a hydroxyapatite.

Ultrasonic root-end preparation with smooth and diamond-coated tips.

The purpose of this study was to compare the effects of smooth and diamond-coated ultrasonic retrotips on the external and internal surfaces of root-end preparations with the aid of a scanning electron microscope (SEM). Forty-four mesial roots of human mandibular molars were selected. The canals were cleaned, shaped and obturated using gutta-percha and sealer. The apical portions were resected at a 45 degrees-angle bevel exposing both mesial canals and the isthmus area. The roots were then divided into two groups according to the type of root-end preparation: Group A--performed with smooth retrotips (S) and Group B--performed with diamond-coated retrotips (DC). The specimens were coded and prepared for SEM evaluation. Observations of the external surface preparation showed that the S and DC retrotips produced very well-centered cavities involving both canals and isthmus area with minimal deviations and no perforative defects. When the internal surface of the root-end preparations was evaluated, it was evident that the use of S retrotips resulted in clean canal walls with little superficial debris and smear layer. Internal canal surfaces done with DC retrotips were irregular showing patent grooves, in contrast with the more uniform, regular and smoother surfaces when S retrotips were employed.

In human hepatocellular carcinoma cells the total membrane surface area of each major organelle is a particular allometric function of the cytoplasmic volume. A morphometric study.

Morphometric analysis by both light and electron microscopy was performed in cells from five cases of human, hepatocellular carcinoma (HCC) and in three control cases. In each case, three fragments were examined individually and the following morphometric parameters evaluated: a) nuclear, cytoplasmic and cell volumes; b) volume density and absolute volume of the rough ER, smooth ER, mitochondria, Golgi apparatus, peroxisomes, dense bodies and cytoplasmic matrix; c) surface density, surface/ volume ratio, and total surface area of rough ER, smooth ER and outer mitochondrial membranes. The parameters obtained from HCC cases showed ample scatter of data, all control values lying within the interval between the extreme values for the various parameters. Both the original and the logarithmically transformed data on volume and total membrane surface area of organelles (y) and of the cytoplasmic volume (x) were regressed using first degree regression equations. The original values for volume and total surface area of rough ER, total ER and mitochondria were linearly related to the corresponding values for cytoplasmic volume. The allometric analysis carried out with the logarithms also revealed significant regressions between cytoplasmic volume and smooth ER parameters not detectable when using the original x and y values. It showed, in addition, that in progressively larger cytoplasmic volumes, the cisternae of both rough and smooth ER tend to appear more compacted and a higher portion of the total ER membrane tends to be constituted of smooth ER. Within the wide range of variation in cytoplasmic volume of the HCC cells, the volume and total surface of the organelles do not vary randomly. These data indicate that in the small, normal-sized and large tumoral cells the mechanisms responsible for the cytoplasmic volume and for the corresponding total volume and membrane surface area of each major organelle are interdependent. Such an interdependence gives no support to ideas implying that the variation in size of cancer cells, an element of pleomorphism, would result of anarchical intracellular synthetic and/or degradative conditions.

A three-dimensional reconstruction study of the rough ER-Golgi interface in serial thin sections of the pancreatic acinar cell of the rat.

Distinctive views of the tubulo-vesicular elements interposed between the endoplasmic reticulum (ER) and the Golgi apparatus were obtained in thin sections. The tubules that protrude from the transitional rough ER (tRER) are of dissimilar length. The numbers of tubules and of the nearby omega- and pear-shaped profiles decrease after fasting and are partially restored by refeeding. This formation is designated herein as the budding chamber of the tRER. Close to the budding chamber, clusters of 56 nm diameter vesicles are consistently observed. In some of the cells, convoluted tubules appear enmeshed with the presumptive transport vesicles of 56 nm diameter and with irregular, vesicular formations. Apparently structureless, electron-lucent ellipsoidal areas are found adjacent to these membranous elements. Serial and semi-serial sections show that the budding chamber, the sinuous tubules, the irregular vesicles, the structureless regions and the 56 nm vesicles fill tunnel-like spaces limited by the outermost Golgi cisterna (OGC) on one side and by the tRER on the other. Curved tubules appear to link the lumen of the OGC with that of smooth membranous occupants of these tunnel-like spaces. A presumptive luminal connection between these membranous occupants and the tubules of the budding chamber can also be seen. The predominant configuration of the OGC is that of a perforated, flat saccule. However, OGC regions exhibiting progressively lower densities of fenestrae, including smooth surfaced sectors eventually accumulating an intraluminal content are seen. Two such dilated, saccular portions of the OGC were analyzed through reconstruction of serial sections. Bundles of microtubules run closely apposed to the cis side of the OGC.

Development of the cloacal bursa in the domestic fowl. II. A quantitative and fine structural analysis of the follicular cortex and medulla.

The estimated volumes of the follicular medulla (x) and cortex (y) from 14-day-old embryos till 28-day-old White Leghorn chicks were associated through the allometric formula y = bxk or log y = log b + k log x. Two successive allometric growth stages (I and II) are discernable, being hatching the transition region between them. The volumetric growth of the cortex is 2.49 times greater than that of the medulla in stage I, whereas cortex and medulla grow isometrically in stage II. The curve fitting procedure analysis of the absolute cortical and medullary growth confirmed these results. The fine structure of the cell types in the follicular medulla revealed that: a) in the allometric stage I pre-existing, bud precursor (Pr) cells appear to give rise to basal (Ba) and medullary epithelial (ME) cells, in both cases showing lucent and dark varieties. A medullary cytoreticulum is established at the onset of this stage. b) The marked lymphocyte proliferation during stage II occurs among the thin and short cytoplasmic processes of BA cells. These processes extend towards the centre of the medulla and also show many lateral interdigitating processes. During this same stage, the cytoplasmic processes of ME cells elongate and become thinner promoting a widening of the cytoreticulum interstices. The fine structural analysis of the cortical cytoarchitectural arrangement showed that: a) before the onset of stage I (14-day-old embryos) the cortex consists mainly of typical fibroblasts (F) and a few blastic (Pr?) cells. Later in this stage I, a poorly defined cortical framework is made up of typical fibroblasts, few cortical branching (CB) cells of the epithelial variety (which seem to be derived from Pr cells) and CB cells of the fibroblast-like variety. These cells are interspersed with mature and immature lymphocytes. b) Allometric stage II of the cortex is characterized by the presence of very thin and long cytoplasmic processes from CB cells of both epithelial and fibroblast-like varieties. The arrangement of CB cell profiles, visualized in electron micrographic montages, is remarkably similar to that of the ME cells profiles which are known to form a cytoreticulum. We thus propose that the mature follicular cortex is endowed with a cellular framework forming wide interstices in which packed mature lymphoid cells are lodged.

Morphometric studies on venom secretory cells from Bothrops jararacussu (Jararacuçu) before and after venom extraction.

A comparative morphometrical analysis was carried out on secretory cells from Bothrops jararacussu venom glands, before manual extraction of the venom (milking) and 4 and 8 days after milking. At the 8th day after milking, the cytoplasmic volume increased by 160%. The rough endoplasmic reticulum (RER) volume density increase, up to the 8th day after milking, is mainly due to widening of the intra-scisternal space. The total volume and membrane surface of the RER. Golgi apparatus and subcomponents, secretory vesicles and mitochondria, increased during the experimental period while the volume and surface densities of these organelles, with the exception of the RER, did not vary. The numerical density of Golgi-associated microvesicles per Golgi volume unit also increased. The greatest relative increments in these parameters occurred within the first 4 days. These results are compatible with an increased rate of membrane synthesis and transport in the milked glands and suggest that the membrane biogenesis, degradation and circulation that takes place in the first week after milking is achieved through coordinated cellular mechanisms that maintain the rate between total membrane surface and total cytoplasmic volume unaltered.