Effect of Calcium on the in vitro Growth of Aechmea blanchetiana (Baker) L. B. Smith Plantlets

This work investigated the influence of different concentrations of calcium on the growth of plantlets of the bromeliad Aechmea blanchetiana cultured in vitro. Seedlings of A. blanchetiana were axenically cultured in liquid Murashige and Skoog basal medium supplemented with different concentrations of calcium (Ca; 1.5, 3, 4.5, 6, or 12 mM) without growth regulators. The resulting plantlets were cultured under 93 mol m-2 s-1 illumination, 12 hour photoperiod regime and 25C 1 for 120 days with subculture to fresh identical media every 30 days. The addition of calcium at 9.38 mM to MS modified medium increased the production of fresh and dry mass of plantlets, whilst chlorine from calcium chloride dehydrate (CaCl2 2 H2O) in excess (3.35 mM) decreased both the fresh and dry mass of plantlets.

Effect of cooking on non-starch polysaccharides of hard-to-cook beans

Experiments carried out to study changes induced by hard-to-cook (HTC) phenomenon in the non-starch polysaccharides of beans stored at 30 degrees C and 75% RH for 8 months showed that the development of HTC did not affect the amounts of soluble and insoluble fibre in cooked seeds but changed their carbohydrates physical properties. Aged beans non-starch polysaccharides presented lower water-solubility and underwent lower degradation of galacturonans and arabinose-rich polysaccharides when submitted to cooking. The decrease in non-starch polysaccharides water-solubility produced a shift in the polymers fractionation profile which resulted in an increase of weak and middle-alkali soluble polymers bulk as well as in their arabinose and uronic acid contents. Uronic acid contents were higher in polymers released by 1 M NaOH and in the cellulose-rich residues while the arabinose contents were higher in the mild-alkali soluble polymers of aged seeds. Methylation analysis showed no evident alterations in the xyloglucans and arabinans branching degree with beans ageing. However, both, the molecular mass of water-soluble pectins and CDTA-soluble pectins, increased. Even though changes in the non-starch polysaccharide solubility were not related to the decrease in the arabinan and xyloglucan degree of branching they may be related to the formation of new chemical interactions other than hydrogen bond. There was a correlation between acidic and neutral polysaccharides insolubilisation in beans ageing and probably in beans hardening. After processing, aged seeds present higher amounts of insoluble fibre when compared to normal beans. (C) 2008 Elsevier Ltd. All rights reserved.

Gene expression analysis of Paracoccidioides brasiliensis transition from conidium to yeast cell

Paracoccidioides brasiliensis infectious process relies on the initial expression of virulence faactors that are assumed to be controlled by molecular mechanisms through which the conidia and/or mycelial fragments convert to yeast cells. In order to analyze the profile of the thermally-induced dimorphic gene expression, 48 h C-L transition cultures which had been incubated at 36 degrees C were studied. By this time approximately 50% of the conidial population had already reverted to yeast form cells. At this transition time, an EST-Orestes library was constructed and characterized. As a result, 79 sequences were obtained, of which 39 (49.4%) had not been described previously in other libraries of this fungus and which could represent novel exclusive C-Y transition genes. Two of these sequences are, among others, cholestanol delta-isomerase, and electron transfer flavoprotein-ubiquinoneoxidoreductase (ETF-QO). The other 40 (50.6%) sequences were shared with Mycelia (M), Yeast (Y) or Mycelia to yest transition (M-Y) libraries. An important component of this group of sequences is a putative response regulator receiver SKN7, a protein of high importance in stress adaptation and a regulator of virulence in some bacteria and fungi. This is the first report identifying genes expressed during the C-Y transition process, the initial step required to understand the natural history of P brasiliensis conidia induced infection.

Phenotypic analysis of genes whose mRNA accumulation is dependent on calcineurin in Aspergillus fumigatus

Calcineurin plays an important role in the control of cell morphology and virulence in fungi. Calcineurin is a serine/threonine-specific protein phosphatase heterodimer consisting of a catalytic subunit A and a regulatory subunit B. A mutant of Aspergillus fumigatus lacking the calcineurin A (calA) catalytic subunit exhibited defective hyphal morphology related to apical extension and branching growth, which resulted in drastically decreased filamentation. Here, we investigated which pathways are influenced by A. fumigatus calcineurin during proliferation by comparatively determining the transcriptional profile of A. fumigatus wild type and Delta calA mutant strains. Our results showed that the mitochondrial copy number is reduced in the Delta calA mutant strain, and the mutant has increased alternative oxidase (aoxA) mRNA accumulation and activity. Furthermore, we identified four genes that encode transcription factors that have increased mRNA expression in the Delta calA mutant. Deletion mutants for these transcription factors had reduced susceptibility to itraconazole, caspofungin, and sodium dodecyl sulfate (SDS). (C) 2009 Elsevier Inc. All rights reserved.

Recurrent Laryngeal Nerve A Plexus Rather Than a Nerve?

Objectives: To analyze the frequency of extralaryngeal branching (ELB) of the recurrent laryngeal nerve (RLN) in a consecutive series of patients undergoing thyroidectomy by the same group of surgeons during an extended period and to compare our findings with the data available in the literature. Design: Retrospective medical record study. Setting: Academic research. Patients: From March 1, 1983, to September 30, 2008, 2677 patients underwent thyroidectomy. Of these, 1638 patients had surgical information about at least 1 RLN. A total of 1081 patients underwent bilateral operations. During the last 5 years of the study, intraoperative laryngeal nerve monitoring was performed in selected patients using a commercially available system. Main Outcome Measures: Information was obtained regarding 2154 RLNs. Results: A total of 1390 RLNs (64.53%) had ELB. Among 447 patients in whom intraoperative laryngeal nerve monitoring was used, the anterior branches usually exhibited more electrophysiologic activity. Conclusions: Extralaryngeal branching was found in 64.53% of RLNs in this case series. In recent patients with intraoperative laryngeal nerve monitoring, electrophysiologic activity was observed in the branches, particularly the anteriorly situated ones. Recognition of this frequent anatomical configuration and meticulous preservation of all branches are of paramount importance to decrease postoperative morbidity associated with thyroidectomy.

Asbestos-related Disease Progression of Parenchymal Abnormalities on High-resolution CT

Purpose: To evaluate the changes over time in the pattern and extent of parenchymal abnormalities in asbestos-exposed workers after cessation of exposure and to compare 3 proposed semiquantitative methods with a careful side-by-side comparison of the initial and the follow-Lip computed tomography (CT) images. Materials and Methods: The study included 52 male asbestos workers (mean age SD, 62.2y +/- 8.2) who had baseline high-resolution CT after cessation of exposure and follow-up CT 3 to 5 years later. Two independent thoracic radiologists quantified the findings according to the scoring systems proposed by Huuskonen, Gamsu, and Sette and then did a side-by-side comparison of the 2 sets of scans without awareness of the dates of the CT scans. Results: There was no difference in the prevalence of the 2 most common parenchymal abnormalities (centrilobular small dotlike or branching opacities and interstitial lines) between the initial and follow-up CT scans. Honeycombing (20%) and traction bronchiectasis and bronchiolectasis (50%) were seen more commonly on the follow-up CT than on the initial examination (10% and 33%, respectively) (P = 0.01). Increased extent of parenchymal abnormalities was evident on side-by-side comparison in 42 (81%) patients but resulted in an increase in score in at least 1 semiquantitative system in only 16 (31%) patients (all P > 0.01, signed test). Conclusions: The majority of patients with previous asbestos exposure show evidence of progression of disease on CT at 3 to 5 years follow-up but this progression is usually not detected by the 3 proposed semiquantitative scoring schemes.

Prognostic relevance of TTF-1 and MMP-9 expression in advanced lung adenocarcinoma

Background: The thyroid transcription factor-1 (TTF-1) is a tissue-specific transcription factor that Could playan important role in cell differentiation and morphogenesis of lung tumors. Matrix metalloproteinase-9 (MMP-9) is a protease commonly expressed in non-small cell lung cancer, conferring angiogenic and metastatic potential. Methods: We assessed TTF-1 and MMP-9 tumor expression by immunohistochemistry in 51 patients with lung adenocarcinoma, stage 11113 or IV, treated with platinum regimens. A bicategorical prognostic model was obtained using the Kaplan-Meier method, COX regression, and conjunctive consolidation. Results: The median expression of TTF-1 was 30.0% (range: 0-85.9%). All tumors expressed MMP-9 (median: 78.7%: range: 15.2-96.1%). Median survival was 41.6 weeks, with estimated 1- and 2-year survival rates of 45.0% and 22.0%, respectively. Poor performance status (Karnofsky scale) - hazards ratio(HR): 1.03. 95% confidence interval (CI): 1.01-1.06: low TTF-1 expression (<40%) - FIR: 4.00, 95% CI: 1.75-9.09: and high MMP-9 expression (>= 80%) - HR: 2.82, 95% CI: 1.30-6.08 were independent prognostic factors. Patients could be stratified in three death risk groups according to markers expression: low risk (high TTF-1 and low MMP-9; median survival: 127.6 weeks), intermediate risk (low TTF-1 OF high MMP-9; median survival: 39.0 weeks): and high risk (low TTF-1 and high MMP-9: median survival: 16.4 weeks). Conclusion: TTF-1 and MMP-9 tumor expression as detected by immunohistochemistry may allow identification of different, clinically meaningful, prognostic groups of advanced lung adenocarcinoma patients treated with platinum regimens. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Down-regulation of the candidate tumor suppressor gene PAR-4 is associated with poor prognosis in breast cancer

Substantial experimental evidence indicates that PAWR gene (PKC apoptosis WT1 regulator; also named PAR-4, prostate apoptosis response-4) is a central player in cancer cell survival and a potential target for cancer-selective targeted therapeutics. However, little is known about the role of PAR-4 in breast cancer. We investigated the possible role of PAR-4 expression in breast cancer. IHC results on tissue microarrays containing 1,161 primary breast tumor samples showed that 57% (571/995) of analyzable cases were negative for PAR-4 nuclear staining. Down-regulation of nuclear PAR-4 protein expression predicted a poor prognosis for breast cancer patients (OS; P=0.041, log-rank test). PAR-4 down-regulation also correlates with poor survival in the group of patients with luminal A subtype breast cancer (P=0.028). Additionally, in this large series of breast cancer patients, we show that ERBB2/HER2, EGFR and pAKT protein expression are significantly associated with shorter disease-free survival and overall survival, but the prognosis was even worse for HER2-positive, EGFR-positive or pAKT-positive breast cancer patients with tumors negative for nuclear PAR-4 expression. Furthermore, using three-dimensional (3D) cell culture we provide preliminary results showing that PAR-4 is highly expressed in the MCF10A cells inside the acini structure, suggesting that PAR-4 might have a role in the lumen acini formation. Taken together, our results provide, for the first time, evidence that PAR-4 may have a role in the process of the mammary eland morphogenesis and its functional inactivation is associated with tumor aggressive phenotype and might represent an additional prognostic and predictive marker for breast cancer.

Descriptive findings on the morphology of dendritic spines in the rat medial amygdala

The rat posterodorsal medial amygdala (MePD) is a brain area in which gonadal hormones induce notable plastic effects in the density of dendritic spines. Dendritic spines are post-synaptic specializations whose shape and spacing change neuronal excitability. Our aim was to obtain new data on the dendritic spines morphology and density from MePD neurons using the carbocyanine dye Dil under confocal microscopy. In adult male rats, the dendritic spine density of the medial branches of the left MePD (mean +/- SD) was 1.15 +/- 0.67 spines/dendritic mu m. From the total sampled, approximately 53% of the spines were classified as thin, 22.5% as ""mushroom-like"", and 21.5% as stubby/wide. Other spine shapes (3%) included those ramified, with a filopodium-like or a gemule appearance, and others with a protruding spinule. Additional experiment joining Dil and synaptophysin (a pre-synaptic protein) labeling suggested synaptic sites on dendritic shafts and spines. Dendritic spines showed synaptophysin puncta close to their head and neck, although some spines had no evident labeled puncta on them or, conversely, multiple puncta appeared upon one spine. These results advance previous light microscopy results by revealing features and complexities of the dendritic spines at the same time that give new insight on the possible synaptic organization of the adult rat MePD. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

The ""single-section"" Golgi method adapted for formalin-fixed human brain and light microscopy

The Golgi method has been used for over a century to describe the general morphology of neurons in the nervous system of different species. The ""single-section"" Golgi method of Gabbott and Somogyi (1984) and the modifications made by Izzo et al. (1987) are able to produce consistent results. Here, we describe procedures to show cortical and subcortical neurons of human brains immersed in formalin for months or even years. The tissue was sliced with a vibratome, post-fixed in a combination of paraformaldehyde and picric acid in phosphate buffer, followed by osmium tetroxide and potassium dicromate, ""sandwiched"" between cover slips, and immersed in silver nitrate. The whole procedure takes between 5 and 11 days to achieve good results. The Golgi method has its characteristic pitfalls but, with this procedure, neurons and glia appear well-impregnated, allowing qualitative and quantitative studies under light microscopy. This contribution adds to the basic techniques for the study of human nervous tissue with the same advantages described for the ""single-section"" Golgi method in other species; it is easy and fast, requires minimal equipment, and provides consistent results. (C) 2010 Elsevier B.V. All rights reserved.

Loss-of-function mutations in the genes encoding prokineticin-2 or prokineticin receptor-2 cause autosomal recessive Kallmann syndrome

Context: Physiological activation of the prokineticin pathway has a critical role in olfactory bulb morphogenesis and GnRH secretion in mice. Objective: To investigate PROK2 and PROKR2 mutations in patients with hypogonadotropic hypogonadism (HH) associated or not with olfactory abnormalities. Design: We studied 107 Brazilian patients with HH (63 with Kallmann syndrome and 44 with normosmic HH) and 100 control individuals. The coding regions of PROK2 and PROKR2 were amplified by PCR followed by direct automatic sequencing. Results: In PROK2, two known frameshift mutations were identified. Two brothers with Kallmann syndrome harbored the homozygous p. G100fsX121 mutation, whereas one male with normosmic HH harbored the heterozygous p. I55fsX56 mutation. In PROKR2, four distinct mutations (p. R80C, p. Y140X, p. L173R, and p. R268C) were identified in five patients with Kallmann syndrome and in one patient with normosmic HH. These mutations were not found in the control group. The p. R80C, p. L173R, and p. R268C missense mutations were identified in the heterozygous state in the HH patients and in their asymptomatic first-degree relatives. In addition, nomutations of FGFR1, KAL1, GnRHR, KiSS-1, or GPR54 were identified in these patients. Notably, the new nonsense mutation (p. Y140X) was identified in the homozygous state in an anosmic boy with micropenis, bilateral cryptorchidism, and high-arched palate. His asymptomatic parents were heterozygous for this severe defect. Conclusion: We expanded the repertoire of PROK2 and PROKR2 mutations in patients with HH. In addition, we show that PROKR2 haploinsufficiency is not sufficient to cause Kallmann syndrome or normosmic HH, whereas homozygous loss-of-function mutations either in PROKR2 or PROK2 are sufficient to cause disease phenotype, in accordance with the Prokr2 and Prok2 knockout mouse models.

Immunoprofile of Kuttner tumor (chronic sclerosing sialadenitis)

In the present study, the immunoprofile of chronic sclerosing sialadenitis, also known as Kuttner tumor, was analyzed. Two,cases that occurred in the submandibular gland of male patients were submitted to immunohistochemical reactions to different antibodies. Histological examinations showed a submandibular gland exhibiting various degrees of atrophy with destruction of acini, infiltration by inflammatory cells, and periductal fibrosis. Reactions to cytokeratins (CKs) showed acini and duct remnants positive to CKs 7, 8, 19, and 13. CK14 stained myoepithelial cells around preserved acini and intercalated duct, and also basal cell of excretory ducts, but was negative in proliferating and branching ducts. Smooth muscle actin (SMA) was expressed by myofibroblasts in periductal fibrosis, and an intense expression of extracellular components was also seen. Lymphocyte markers showed, besides mature follicles, a higher presence of CD45RO positive cells. Thus, the immunoprofile of Kuttner is much more in keeping with an inflammatory-induced degenerative disease than with a preneoplastic lesion.

Expression of transforming growth factor-beta 1, -beta 2, and -beta 3 in human developing teeth: Immunolocalization according to the odontogenesis phases

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that has several biological effects in vivo including control of cell growth and differentiation, cell migration, lineage determination, motility, adhesion, apoptosis, and synthesis and degradation of extracellular matrix, and TGF-beta plays an important role in regulating tissue repair and regeneration. Our study analyzed the participation of TGF-beta 1, -beta 2, and -beta 3 in the different stages of morphogenesis and differentiation of human developing dental organ using immunobistochemistry. The maxillae and mandibles of 10 human embryos ranging from 8 to 23 weeks of gestation were employed, according to the approval of the ethical committee. Our study revealed that the TGF-beta subunits-beta 1, beta 2, and beta 3 were present in the various stages of tooth development, but the expression varied according to the differentiation stage, tissue, and TGF-beta subunit. Our results indicated that TGF-beta 1 is closely related to differentiation of enamel organ and initiation of matrix secretion, TGF-beta 2 to cellular differentiation, and TGF-beta 3 to mineral maturation matrix.

Insights from Studies with Oral Cleft Genes Suggest Associations between WNT-pathway Genes and Risk of Oral Cancer

Oral squamous cell carcinoma (OSCC) accounts for more than 90% of the malignant neoplasms that arise in the mucosa of the upper aerodigestive tract. Recent studies of cleft lip/palate have shown the association of genes involved in cancer. WNT pathway genes have been associated with several types of cancer and recently with cleft lip/palate. To investigate if genes associated with cleft lip/palate were also associated with oral cancer, we genotyped 188 individuals with OSCC and 225 control individuals for markers in AXIN2, AXIN1, GSK3 beta, WNT3A, WNT5A, WNT8A, WNT11, WNT3, and WNT9B. Statistical analysis was performed with PLINK 1.06 software to test for differences in allele frequencies of each polymorphism between cases and controls. We found association of SNPs in GSK3B (p = 0.0008) and WNT11 (p = 0.03) with OSCC. We also found overtransmission of GSK3B haplotypes in OSCC cases. Expression analyses showed up-regulation of WNT3A, GSK3B, and AXIN1 and down-regulation of WNT11 in OSCC in comparison with control tissues (P < 0.001). Additional studies should focus on the identification of potentially functional variants in these genes as contributors to human clefting and oral cancer.

Deficiency of the Cytoskeletal Protein SPECC1L Leads to Oblique Facial Clefting

Genetic mutations responsible for oblique facial clefts (ObFC), a unique class of facial malformations, are largely unknown. We show that loss-of-function mutations in SPECC1L. are pathogenic for this human developmental disorder and that SPECC1L is a critical organizer of vertebrate facial morphogenesis. During murine embryogenesis, Speed 1 1 is expressed in cell populations of the developing facial primordial, which proliferate and fuse to form the face. In zebrafish, knockdown of a SPECC1L homolog produces a faceless phenotype with loss of jaw and facial structures, and knockdown in Drosophila phenocopies mutants in the integrin signaling pathway that exhibit cell-migration and -adhesion defects. Furthermore, in mammalian cells, SPECC1L colocalizes with both tubulin and actin, and its deficiency results in defective actin-cytoskeleton reorganization, as well as abnormal cell adhesion and migration. Collectively, these data demonstrate that SPECC1L functions in actin-cytoskeleton reorganization and is required for proper facial morphogenesis.