Morphologic characterisation of the posterior inferior cerebellar artery. A direct anatomic study.

BACKGROUND: The number of studies on the cerebellar arteries has increased. The purpose of this study was to determine the morphological expression of posterior inferior cerebellar artery in a sample of Colombian population. MATERIALS AND METHODS: One hundred eighty-six posterior inferior cerebellar arteries of fresh cadavers were studied. In each specimen, vertebral arteries were injected with 100 mL of semi-synthetic resin, dyed with mineral red. RESULTS: In the 93 blocks of brainstem and cerebellum evaluated, 174 (93.5%) posterior inferior cerebellar arteries were found. Also, there were 12 (6.5%) ageneses. There was single posterior inferior cerebellar artery in 159 (91.4%) samples and duplicate in 10 (5.7%), while 5 (2.9%) specimens showed hypoplasia. The posterior inferior cerebellar artery originated from the vertebral artery in 121 (69.5%) samples and from the basilar artery in 42 (24.1%) samples; while in 11 (6.4%) it originated in a common trunk with the anterior inferior cerebellar artery. In 101 (83.5%) cases, the posterior inferior cerebellar artery originated from the intracranial segment of the vertebral artery, while 20 (16.5%) samples originated from the extracranial segment. The calibres of posterior inferior cerebellar artery in its proximal and distal segments were 1.45 ± 0.37 mm and 1.33 ± 0.31 mm, respectively. CONCLUSIONS: This study, carried out in cadaveric material, provides relevant qualitative and morphometric information of the posterior inferior cerebellar artery, useful for the diagnosis and clinical management, as well as for the surgical approaches that may compromise this structure.

Morphometric analysis of cardiac conduction fibers in horses and dogs, a comparative histological and immunohistochemical study with findings in human hearts.

The principal function of the ventricular conduction system is rapid electrical activation of the ventricles. The aim of this study is to conduct a morphometric study to pinpoint the morphological parameters that define cardiac conduction cells, allowing us to distinguish them from other cells. Five male horse hearts and five male dog hearts were used in the study. The hearts were fixed in a 5% formaldehyde solution. Histological sections of 5 µm thickness were acquired and stained with hematoxylin-eosin and Masson's trichrome and cardiac conduction cells and their junctions were identified by desmin, connexin 40 and a PAS method. We found statistically significant differences in cardiac conduction fibers density and thickness, which was much higher in horses than in dogs (p = 0.000 for both values). By comparing the measured parameters of the cells in both species, we determined that cardiac conduction cells area and diameters were greater in horses than in dogs (p = 0.000 for all values). In dogs there are more junctions (30.8%) than in horses (26.1%), a statistically significant difference (p = 0.041). Our findings regarding the cardiac conduction fibers distribution in the animal species studied becomes new knowledge that contributes to the morphological study of this component of the cardiac conduction system and also makes it possible to locate exactly the site with the highest density of cardiac conduction fibers as a contribution to the cardiological study of these structures that lead to the prevention of ventricular arrhythmias and the identification of their treatment site.

Morphometry and comparative histology of sinus and atrioventricular nodes in humans and pigs and their relevance in the prevention of nodal arrhythmias.

The cardiac conduction system is a network structure that allows the initiation and fast propagation of electrical impulses that trigger the electrical depolarization of the myocardial tissue. The purpose of this work is to study the histological and morphometric characteristics of the different components of the sinus and atrioventricular nodes in humans and pigs and their relationship with supraventricular arrhythmias. In this study, we describe the morphometry of the sinus and atrioventricular nodes of 10 adult humans and 10 pig hearts. A computerized morphometric study has been carried out, where we determined the number of cells that compose the nodes as well as different parameters related to their shape and size. The sinus node in human and pig is a compact structure, whose shape is oblong. Their cells (nodal and transitional cells) are pale and located in the center and the periphery, respectively. The atrioventricular node has also a shape oblong. P cells are pale in both species, but in humans, they are smaller than cardiomyocytes. The T cells are small and pale in both species, identified by hematoxylin-eosin and desmin stains. We have observed through a morphometric profile that the structure of sinus and atrioventricular nodes of pigs and humans show few differences. Pigs can be used as models for hemodynamic applications and experimental studies that include atrial electrical conduction and, in this way, prevent the presentation of arrhythmias that can generate sudden deaths in humans and pigs.

Histological and morphometric study of the components of the sinus and atrioventricular nodes in horses and dogs.

The cardiac nodes are the source of the electrical impulse that is transmitted to the heart, the aim of this work is study the histological and morphometric characteristics of the different components of the sinus and atrioventricular nodes in horses and dogs that help to know the physiopathology of these nodes. A group of ten horse hearts and five dog hearts were used. The region of the sinus and atrioventricular nodes was sectioned serially, and the block of tissue removed for study. The samples were assessed using a morphometric analysis with the Image-Pro Plus 7.1 software and the acquisition of the images using a Leica DMD108 optic microscope. The shape of the horse's sinus node is oblong and its P cells are large. The shape of the dog's sinus is rounded or oblong. The P cells are large and pale. The area of P cells in horses was 976 (SD 223.7) µm2 and in dogs the area for P cells was 106 (SD 30.4) µm2, which indicates that the value for P cells in horses are significantly higher than in dogs (p = .001). The horse atrioventricular node presented an oblong shape and in dogs, presents a spindle shape. The lower cell density in any of the cardiac nodes, especially in P cells of sinus node, can decrease electrical conduction within the nodes and in the internodal tracts, which would reflect the presence of cardiac arrhythmias derived from poor conduction, even in morphologically normal hearts.

Insertion of the LINE-1 element in the C-MYC gene and immunoreactivity of C-MYC, p53, p21 and p27 proteins in different morphological patterns of the canine TVT / Inserção do elemento LINE - 1 no gene C-MYC e imunorreatividade das proteinas C-MYC, p53, p21 e p27 nos diferentes padrões morfológicos do tumor TVT

The canine transmissible venereal tumor (TVT) affects the external genitalia of dogs by the natural transplant of viable tumor cells. Thus, this research aimed to diagnose and characterize TVT morphological patterns, identify the insertion of the LINE-1 element in C-MYC gene, by means of the polymerase chain reaction (PCR), and evaluate the immunohistochemical expression of C-MYC, p53, p21 and p27 proteins. The relationship between C-MYC and p53 proteins and their interference on the expression of p21 and p27 were also studied. For that, 20 samples of naturally occurring TVT were used, subjected to cytopathological, histopathological and immunohistochemical analysis, and to molecular diagnosis of neoplasia. The increased tissue expression and the correlation among C-MYC, p53, p21 and p27 proteins indicate reduction and/or loss of their functionality in the TVT microenvironment, with consequent apoptotic suppression, maintenance of cell growth and progression of neoplasia.(AU)

O tumor venéreo transmissível canino (TVT) afeta a genitália externa de cães pelo transplante natural de células tumorais viáveis. Assim, esta pesquisa teve como objetivo diagnosticar e caracterizar TVT em padrões morfológicos, identificar a inserção do elemento LINE-1 em gene C-MYC, por meio da reação em cadeia da polimerase (PCR), e avaliar a expressão imuno-histoquímica do C-MYC, p53, p21 e p27. A relação entre C-MYC e as proteínas p53 e a sua interferência na expressão de p21 e p27 foram também estudadas. Para isso, foram utilizadas 20 amostras de ocorrência natural de TVT, submetido a exame citopatológico, histopatológica e imuno-histoquímica e ao diagnóstico molecular de neoplasia. A expressão aumentada do tecido e a correlação entre a C-MYC e as proteínas p53, p21 e p27 indicam redução e/ou perda de funcionalidade na TVT em seu microambiente, com consequente supressão apoptótica, manutenção do crescimento celular e progressão da neoplasia.(AU)

Development of an IP-Free Biotechnology Platform for Constitutive Production of HPV16 L1 Capsid Protein Using the Pichia pastoris PGK1 Promoter.

The human papillomavirus (HPV) L1 major capsid protein, which forms the basis of the currently available vaccines against cervical cancer, self-assembles into virus-like particles (VLPs) when expressed heterologously. We report the development of a biotechnology platform for HPV16 L1 protein expression based on the constitutive PGK1 promoter (PPGK1) from the methylotrophic yeast Pichia pastoris. The L1 gene was cloned under regulation of PPGK1 into pPGKΔ3 expression vector to achieve intracellular expression. In parallel, secretion of the L1 protein was obtained through the use of an alternative vector called pPGKΔ3α, in which a codon optimized α-factor signal sequence was inserted. We devised a work-flow based on the detection of the L1 protein by dot blot, colony blot, and western blot to classify the positive clones. Finally, intracellular HPV VLPs assembly was demonstrated for the first time in yeast cells. This study opens up perspectives for the establishment of an innovative platform for the production of HPV VLPs or other viral antigens for vaccination purposes, based on constitutive expression in P. pastoris.

Near-self-imaging cavity for three-mode optoacoustic parametric amplifiers using silicon microresonators.

Three-mode optoacoustic parametric amplifiers (OAPAs), in which a pair of photon modes are strongly coupled to an acoustic mode, provide a general platform for investigating self-cooling, parametric instability and very sensitive transducers. Their realization requires an optical cavity with tunable transverse modes and a high quality-factor mirror resonator. This paper presents the design of a table-top OAPA based on a near-self-imaging cavity design, using a silicon torsional microresonator. The design achieves a tuning coefficient for the optical mode spacing of 2.46 MHz/mm. This allows tuning of the mode spacing between amplification and self-cooling regimes of the OAPA device. Based on demonstrated resonator parameters (frequencies ∼400 kHz and quality-factors ∼7.5×10(5) we predict that the OAPA can achieve parametric instability with 1.6 µW of input power and mode cooling by a factor of 1.9×10(4) with 30 mW of input power.

Utilization of glycerin byproduct derived from soybean oil biodiesel as a carbon source for heterologous protein production in Pichia pastoris.

Crude glycerol, also known as glycerin, is the main byproduct of the biodiesel industry. It has been estimated that up to 40,000 tons of glycerin will be produced each year by 2020. This study evaluated the value-added use of crude glycerol derived from soybean biodiesel preparation as a carbon source for heterologous protein production using the yeast Pichia pastoris. Eleven glycerin samples were obtained by methanolysis of soybean oil using different acids or bases as catalysts. Cell growth experiments showed that crude glycerol containing either potassium or sodium hydroxide resulted in 1.5-2 times higher final cell densities when compared to glycerol P.A. Finally, crude glycerol containing sodium hydroxide was successfully utilized for constitutive heterologous α-amylase production in P. pastoris. This study demonstrated that crude glycerol without any purification steps may be directly used as carbon source for protein production in P. pastoris.

Culture of bovine ovarian follicle wall sections maintained the highly estrogenic profile under basal and chemically defined conditions.

Follicle cultures reproduce in vitro the functional features observed in vivo. In a search for an ideal model, we cultured bovine antral follicle wall sections (FWS) in a serum-free defined medium (DM) known to induce 17ß-estradiol (E2) production, and in a nondefined medium (NDM) containing serum. Follicles were sectioned and cultured in NDM or DM for 24 or 48 h. Morphological features were determined by light microscopy. Gene expression of steroidogenic enzymes and follicle-stimulating hormone (FSH) receptor were determined by RT-PCR; progesterone (P4) and E2 concentrations in the media were measured by radioimmunoassay. DM, but not NDM, maintained an FWS morphology in vitro that was similar to fresh tissue. DM also induced an increase in the expression of all steroidogenic enzymes, except FSH receptor, but NDM did not. In both DM and NDM, there was a gradual increase in P4 throughout the culture period; however, P4 concentration was significantly higher in NDM. In both media, E2 concentration was increased at 24 h, followed by a decrease at 48 h. The E2:P4 ratio was higher in DM than in NDM. These results suggest that DM maintains morphological structure, upregulates the expression of steroidogenic enzyme genes, and maintains steroid production with a high E2:P4 ratio in FWS cultures.

Culture of bovine ovarian follicle wall sections maintained the highly estrogenic profile under basal and chemically defined conditions

Follicle cultures reproduce in vitro the functional features observed in vivo. In a search for an ideal model, we cultured bovine antral follicle wall sections (FWS) in a serum-free defined medium (DM) known to induce 17β-estradiol (E2) production, and in a nondefined medium (NDM) containing serum. Follicles were sectioned and cultured in NDM or DM for 24 or 48 h. Morphological features were determined by light microscopy. Gene expression of steroidogenic enzymes and follicle-stimulating hormone (FSH) receptor were determined by RT-PCR; progesterone (P4) and E2 concentrations in the media were measured by radioimmunoassay. DM, but not NDM, maintained an FWS morphology in vitro that was similar to fresh tissue. DM also induced an increase in the expression of all steroidogenic enzymes, except FSH receptor, but NDM did not. In both DM and NDM, there was a gradual increase in P4 throughout the culture period; however, P4 concentration was significantly higher in NDM. In both media, E2 concentration was increased at 24 h, followed by a decrease at 48 h. The E2:P4 ratio was higher in DM than in NDM. These results suggest that DM maintains morphological structure, upregulates the expression of steroidogenic enzyme genes, and maintains steroid production with a high E2:P4 ratio in FWS cultures.

Hydrolytic enzymes in Paracoccidioides brasiliensis--ecological aspects

Paracoccidioides brasiliensis is a thermally dimorphic fungus that causes paracoccidioidomycosis. The yeast form of this pathogen is found in the animal host whereas the mycelial form is recovered from living and non-living organic material. The sole carbon source available in these habitats is represented by polysaccharides from the plant cell wall. Hydrolytic enzymes are necessary to convert these polymers into simple sugars for fungal metabolism. We report on the presence of ortholog genes of hydrolytic enzymes identified in the P. brasiliensis transcriptome and on hydrolytic activities in supernatants of induced P. brasiliensis cultures of mycelium and yeast cells. Enzymatic assays have shown cellulase and xylanase activities, both being higher in mycelium than in the yeast form. Amylase and chitinase activities were detected only in mycelium. Data so far reinforce the idea that mycelial P. brasiliensis is a saprobe.

Cell cycle, DNA replication, repair, and recombination in the dimorphic human pathogenic fungus Paracoccidioides brasiliensis

DNA replication, together with repair mechanisms and cell cycle control, are the most important cellular processes necessary to maintain correct transfer of genetic information to the progeny. These processes are well conserved throughout the Eukarya, and the genes that are involved provide essential information for understanding the life cycle of an organism. We used computational tools for data mining of genes involved in these processes in the pathogenic fungus Paracoccidiodes brasiliensis. Data derived from transcriptome analysis revealed that the cell cycle of this fungus, as well as DNA replication and repair, and the recombination machineries, are highly similar to those of the yeast Saccharomyces cerevisiae. Among orthologs detected in both species, there are genes related to cytoskeleton structure and assembly, chromosome segregation, and cell cycle control genes. We identified at least one representative gene from each step of the initiation of DNA replication. Major players in the process of DNA damage and repair were also identified.

Transcriptome characterization of the dimorphic and pathogenic fungus Paracoccidioides brasiliensis by EST analysis.

Paracoccidioides brasiliensis is a pathogenic fungus that undergoes a temperature-dependent cell morphology change from mycelium (22 degrees C) to yeast (36 degrees C). It is assumed that this morphological transition correlates with the infection of the human host. Our goal was to identify genes expressed in the mycelium (M) and yeast (Y) forms by EST sequencing in order to generate a partial map of the fungus transcriptome. Individual EST sequences were clustered by the CAP3 program and annotated using Blastx similarity analysis and InterPro Scan. Three different databases, GenBank nr, COG (clusters of orthologous groups) and GO (gene ontology) were used for annotation. A total of 3,938 (Y = 1,654 and M = 2,274) ESTs were sequenced and clustered into 597 contigs and 1,563 singlets, making up a total of 2,160 genes, which possibly represent one-quarter of the complete gene repertoire in P. brasiliensis. From this total, 1,040 were successfully annotated and 894 could be classified in 18 functional COG categories as follows: cellular metabolism (44%); information storage and processing (25%); cellular processes-cell division, posttranslational modifications, among others (19%); and genes of unknown functions (12%). Computer analysis enabled us to identify some genes potentially involved in the dimorphic transition and drug resistance. Furthermore, computer subtraction analysis revealed several genes possibly expressed in stage-specific forms of P. brasiliensis. Further analysis of these genes may provide new insights into the pathology and differentiation of P. brasiliensis.

[Clinical and epidemiological study of calcifying chronic pancreatitis in Goiânia, GO, Brazil]. / Estudo clínico-epidemiológico da pancreatite crônica calcificante em Goiânia, GO, Brasil.

The authors studied, prospectively, 102 cases of calcifying chronic pancreatitis in Goiânia, GO, Brazil, from 1985 to 1996. Alcohol was the major cause of calcifying chronic pancreatitis, responsible for 94.1% of the cases. In others 5.9% of the cases, not associated with alcohol, the etiologic diagnosis could not be confirmed. The average age of the patients was 39.8 +/- 9.8 years, and the first symptoms appeared with the average age of male sex in the proportion of 9.2/1. The average of alcoholic beverage ingested was 258 +/- 187.1 g/day of ethanol through an average period of 17.5 +/- years. The major complications found were: chronic diarrhea (malabsorption), pleural effusion, cysts, diabetes mellitus, jaundice and digestive hemorrhage. This study, when compared to others from other Brazilian cities, suggests that there are differences in the natural history of calcifying chronic pancreatitis among several regions of Brazil.

B cell-independent selection of memory T cells after mucosal immunization with Candida albicans.

B cell-deficient mice have normal T cell responses to Ags inoculated systemically; however, it is not known whether they can mount systemic and mucosal T cell responses to Ags through normally B cell-enriched gastrointestinal mucosae. Mucosal colonization of germfree, B cell-deficient J(H)D mice with the pathogenic gastrointestinal fungus, Candida albicans selected splenic CD4+ and CD8+ TCR alphabeta memory T cells, as indicated by 1) increased numbers of splenic CD4+ and CD8+ TCR alphabeta expressing T cells of the CD45RB(low) CD44(high) phenotype, 2) early expansion followed by progressive decrease in the number of splenic CD4+ and CD8+ TCR alphabeta T cells, and 3) concomitant increases in the percentage of apoptosis and proliferation in the latter subsets. Although i.v. challenge of germfree or conventional J(H)D mice with C. albicans did not increase apoptosis or induce changes in the number of splenic memory T cells, i.v. challenge of mucosally immunized germfree J(H)D mice led to further proliferation and expansion of activated splenic CD4+ and CD8+ TCR alphabeta thymic-educated memory T cells, which were first evoked by mucosal immunization. Oral colonization with C. albicans also increased the number of gammadelta and thymic and extrathymic alphabeta T cells in gastrointestinal mucosae. In conclusion, our results are the first strong evidence that thymic and extrathymic T cells participate in mucosal immunity to C. albicans in the absence of B cells; however, CD4+ and thymic-educated CD8+ TCR alphabeta memory subsets evoked by mucosal, but not parenteral (i.v.), challenge contribute to protective immunity to systemic candidiasis.

Importance of beta2-microglobulin in murine resistance to mucosal and systemic candidiasis.

beta2-Microglobulin knockout (beta2m-/-) mice, which lack major histocompatibility complex class I expression and are deficient in CD8alpha/beta T-cell receptor alpha/beta (TcRalpha/beta) T cells, were as resistant to systemic (intravenous) challenge with Candida albicans as immunocompetent controls. Conversely, the beta2m-/- mutant mice were susceptible to systemic candidiasis of endogenous origin despite the induction of C. albicans-specific antibody and cell-mediated immune responses after colonization with a pure culture of C. albicans. Despite some superficial and transient infections of tongues and esophagi (detected by histology) at 1 to 2 weeks after oral colonization and gastric infections (cardia-antrum section) which were observed at 10 to 12 weeks after oral challenge, C. albicans-colonized beta2m-/- mice showed an overall resistance to candidiasis in other mucosal and cutaneous tissues. These data suggest that immune defects that accompany the loss of beta2-microglobulin play an important role in murine resistance to gastric and disseminated candidiasis of endogenous (intestinal tract) origin and that innate immunity and CD4 TcRalpha/beta as well as CD8alpha/alpha TcRalpha/beta (or -gamma/delta) T cells play an important role in resistance to systemic, cutaneous, and nongastric mucosal tissues.

Genetic identification of the site of DNA contact in the yeast heat shock transcription factor.

The heat shock transcription factor (HSF), a trimeric transcription factor, activates the expression of heat shock genes in eukaryotes. We have isolated mutations in the HSF1 gene from Saccharomyces cerevisiae that severely compromise the ability of HSF to bind to its normal binding site, repeats of the module nGAAn. One of these mutations, Q229R, shows a "new specificity" phenotype, in which the protein prefers the mutant sequence nGACn. These results identify the region of HSF that contacts DNA, in complete agreement with the crystal structure of HSF of Kluyveromyces lactis and the nuclear magnetic resonance data from HSF of Drosophila melanogaster. To determine the orientation of the DNA-binding domain on the nGAAn motif, we performed site-specific cross-linking between cysteine residues of single-cysteine substitutions. Cysteines placed at the N terminus of the DNA contact helix formed cross-links readily, while cysteines placed at the C terminus of the helix did not.