Structural, functional and immunological studies on a polymeric bacterial protein

The characterization of proteins from Brucella spp, the causative agent of brucellosis, has been the subject of intensive research. We have described an 18-kDa cytoplasmic protein of Brucella abortus and shown the potential usefulness of this protein as an antigen for the serologic diagnosis of brucellosis. The amino acid sequence of the protein showed a low but significant homology with that of lumazine synthases. Lumazine is an intermediate product in bacterial riboflavin biosynthesis. The recombinant form of the 18-kDa protein (expressed in E. coli) folds like the native Brucella protein and has lumazine-synthase enzymatic activity. Three-dimensional analysis by X-ray crystallography of the homolog Bacillus subtilis lumazine synthase has revealed that the enzyme forms an icosahedral capsid. Recombinant lumazine synthase from B. abortus was crystallized, diffracted X rays to 2.7-Å resolution at room temperature, and the structure successfully solved by molecular replacement procedures. The macromolecular assembly of the enzyme differs from that of the enzyme from B. subtilis. The Brucella enzyme remains pentameric (90 kDa) in its crystallographic form. Nonetheless, the active sites of the two enzymes are virtually identical at the structural level, indicating that inhibitors of these enzymes could be viable pharmaceuticals across a broad species range. We describe the structural reasons for the differences in their quaternary arrangement and also discuss the potential use of this protein as a target for the development of acellular vaccines.

Functional, Social and Emotional Values as Determinants of Environmentally Responsible Media Consumption

The primary purpose of this research is to develop an enhanced understanding of how consumption values influence environmentally responsible consumption of print and digital media. Theoretical elaboration considers the associations of functional, social and emotional consumption values, green consumer segmentation and media consumption. Additionally, the purpose is to identify consumer perceptions of print and digital media’s environmental responsibility. Empirical analysis was based on qualitative interviews with a sample of 20 Finnish consumers categorized in two segments: young adults and middle aged consumers. Primary data collection was conducted through individual, semi-structured interviews. To analyze the respondents’ approach on the topic, the interviews disclosed themes of media consumption, perceived environmental friendliness of media, norms of behavior and consumers’ general consumption patterns. The results implicate functional value dominated the consumption decision-making process both in a general level and in media consumption. In addition to functional value, environmental responsibility does provide consumers with both emotional and social values. Analysis on perceived environmental responsibility of media demonstrated consumers generally perceive digital media as an environmentally responsible alternative because it does not create physical paper waste. Nevertheless, the perceptions of environmental responsibility and media consumption patterns lacked a consistent connection. Though, both theory and empirical results indicated an average consumer lacks a comprehensive understanding of digital and print media’s life-cycle and hence their environmental advantages and disadvantages.

Cloning and characterization of Echinococcus granulosus (Cestode) EgactI and EgactII actin gene promoters and their functional analysis in the NIH3T3 mouse cell line

We report here for the first time the structure and function of a promoter from a cestode. The ability of DNA fragments respectively encompassing the 935-bp and 524-bp regions upstream from the ATG codon from the EgactI and EgactII actin genes of Echinococcus granulosus to promote transcription was studied in the NIH3T3 mouse cell line. The results of transfection assays showed that both regions have strong promoter activity in these cells. The fragments were tested in both orientations and the 524-bp fragment of EgactII presented a bidirectional promoter activity. Deletion analysis of EgactI and EgactII promoters indicated the presence of regulatory regions containing putative silencer elements. These results indicate that both EgactI and EgactII promoters are functional and that the preliminary functional evaluation of E. granulosus and possibly of other cestode promoters can be performed in heterologous cell lines.

Extracellular calcium-sensing receptor: structural and functional features and association with diseases

The recently cloned extracellular calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays an essential role in the regulation of extracellular calcium homeostasis. This receptor is expressed in all tissues related to this control (parathyroid glands, thyroid C-cells, kidneys, intestine and bones) and also in tissues with apparently no role in the maintenance of extracellular calcium levels, such as brain, skin and pancreas. The CaR amino acid sequence is compatible with three major domains: a long and hydrophilic aminoterminal extracellular domain, where most of the activating and inactivating mutations described to date are located and where the dimerization process occurs, and the agonist-binding site is located, a hydrophobic transmembrane domain involved in the signal transduction mechanism from the extracellular domain to its respective G protein, and a carboxyterminal intracellular tail, with a well-established role for cell surface CaR expression and for signal transduction. CaR cloning was immediately followed by the association of genetic human diseases with inactivating and activating CaR mutations: familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism are caused by CaR-inactivating mutations, whereas autosomal dominant hypoparathyroidism is secondary to CaR-activating mutations. Finally, we will comment on the development of drugs that modulate CaR function by either activating (calcimimetic drugs) or antagonizing it (calcilytic drugs), and on their potential therapeutic implications, such as medical control of specific cases of primary and uremic hyperparathyroidism with calcimimetic drugs and a potential treatment for osteoporosis with a calcilytic drug.

Functional differences between two morphologically distinct cell subpopulations within a human colorectal carcinoma cell line

The LISP-I human colorectal adenocarcinoma cell line was isolated from a hepatic metastasis at the Ludwig Institute, São Paulo, SP, Brazil. The objective of the present study was to isolate morphologically different subpopulations within the LISP-I cell line, and characterize some of their behavioral aspects such as adhesion to and migration towards extracellular matrix components, expression of intercellular adhesion molecules and tumorigenicity in vitro. Once isolated, the subpopulations were submitted to adhesion and migration assays on laminin and fibronectin (crucial proteins to invasion and metastasis), as well as to anchorage-independent growth. Two morphologically different subpopulations were isolated: LISP-A10 and LISP-E11. LISP-A10 presents a differentiated epithelial pattern, and LISP-E11 is fibroblastoid, suggesting a poorly differentiated pattern. LISP-A10 expressed the two intercellular adhesion molecules tested, carcinoembryonic antigen (CEA) and desmoglein, while LISP-E11 expressed only low amounts of CEA. On the other hand, adhesion to laminin and fibronectin as well as migration towards these extracellular matrix proteins were higher in LISP-E11, as expected from its poorly differentiated phenotype. Both subpopulations showed anchorage-independent growth on a semi-solid substrate. These results raise the possibility that the heterogeneity found in the LISP-I cell line, which might have contributed to its ability to metastasize, was due to at least two different subpopulations herein identified.

alpha-Smooth muscle actin and proliferating cell nuclear antigen expression in focal segmental glomerulosclerosis: functional and structural parameters of renal disease progression

The aim of the present study was to investigate the expression of alpha-smooth muscle actin (alpha-SM-actin) and proliferating cell nuclear antigen (PCNA) in renal cortex from patients with focal segmental glomerulosclerosis (FSGS) and their correlations with parameters of renal disease progression. We analyzed renal biopsies from 41 patients with idiopathic FSGS and from 14 control individuals. The alpha-SM-actin immunoreaction was evaluated using a score that reflected the changes in the extent and intensity of staining in the glomerular or cortical area. The PCNA reaction was quantified by counting the labeled cells of the glomeruli or renal cortex. The results, reported as median ± percentile (25th; 75th), showed that the alpha-SM-actin scores in the glomeruli and tubulointerstitium from the renal cortex were 2.0 (2.0; 4.0) and 3.0 (3.0; 4.0), respectively, in patients with FSGS, and 0.5 (0.0; 1.0) and 0.0 (0.0; 0.5) in the controls. The number of PCNA-positive cells per glomerulus and graded field of tubulointerstitium from the renal cortex was 0.2 (0.0; 0.4) and 1.1 (0.3; 2.2), respectively, for patients with FSGS, and 0.0 (0.0; 0.5) and 0.0 (0.0; 0.0) for controls. The present data showed an increase of alpha-SM-actin and PCNA expression in glomeruli and renal cortex from FSGS patients. The extent of immunoreaction for alpha-SM-actin in the tubulointerstitial area was correlated with the intensity of proteinuria. However, there was no correlation between the kidney expression of these proteins and the reciprocal of plasma creatinine level or renal fibrosis. These findings suggest that the immunohistochemical alterations may be reversible.

Dietary fiber intake, stool frequency and colonic transit time in chronic functional constipation in children

The objective of the present study was to evaluate associations between fiber intake, colonic transit time and stool frequency. Thirty-eight patients aged 4 to 14 years were submitted to alimentary evaluation and to measurement of colonic transit time. The median fiber intake of the total sample was age + 10.3 g/day. Only 18.4% of the subjects presented a daily dietary fiber intake below the levels recommended by the American Health Foundation. In this group, the median left colonic transit time was shorter than in the group with higher dietary fiber intake (11 vs 17 h, P = 0.067). The correlation between stool frequency and colonic transit time was negative and weak for left colon (r = -0.3, P = 0.04), and negative and moderate for rectosigmoid and total colon (r = -0.5, P<0.001 and r = -0.5, P<0.001, respectively). The stool frequency was lower in the group with slow transit time (0.8 vs 2.3 per week, P = 0.014). In conclusion, most patients with chronic functional constipation had adequate dietary fiber intake. The negative correlation between stool frequency and colonic transit time increased progressively from proximal segments to distal segments of the colon. Patients with normal and prolonged colonic transit time differ in terms of stool frequency.

Is the infertility in hypothyroidism mainly due to ovarian or pituitary functional changes?

The objective of the present study was to examine whether hypothyroidism affects the reproductive system of adult female rats by evaluating ovarian morphology, uterus weight and the changes in serum and pituitary concentrations of prolactin and gonadotropins. Three-month-old female rats were divided into three groups: control (N = 10), hypothyroid (N = 10), treated with 0.05% 6-propyl-2-thiouracil (PTU) in drinking water for 60 days, and T4-treated group (N = 10), receiving daily sc injections of L-thyroxine (0.8 µg/100 g body weight) during the last 10 days of the experiment. At the end of 50 days of hypothyroidism no hypothyroid animal showed a regular cycle, while 71% of controls as well as the T4-treated rats showed regular cycles. Corpora lutea, growing follicles and mature Graafian follicles were found in all ovaries studied. The corpora lutea were smaller in both the hypothyroid and T4-replaced rats. Graafian follicles were found in 72% of controls and only in 34% of hypothyroid and 43% of T4-treated animals. Serum LH, FSH, progesterone and estradiol concentrations did not differ among the three groups. Serum prolactin concentration and the pituitary content of the three hormones studied were higher in the hypothyroid animals compared to control. T4 treatment restored serum prolactin concentration to the level found in controls, but only partially normalized the pituitary content of gonadotropins and prolactin. In conclusion, the morphological changes caused by hypothyroidism can be a consequence of higher prolactin production that can block the secretion and action of gonadotropins, being the main cause of the changes observed.

Effect of race, genetic population structure, and genetic models in two-locus association studies: clustering of functional renin-angiotensin system gene variants in hypertension association studies

Previous genetic association studies have overlooked the potential for biased results when analyzing different population structures in ethnically diverse populations. The purpose of the present study was to quantify this bias in two-locus association studies conducted on an admixtured urban population. We studied the genetic structure distribution of angiotensin-converting enzyme insertion/deletion (ACE I/D) and angiotensinogen methionine/threonine (M/T) polymorphisms in 382 subjects from three subgroups in a highly admixtured urban population. Group I included 150 white subjects; group II, 142 mulatto subjects, and group III, 90 black subjects. We conducted sample size simulation studies using these data in different genetic models of gene action and interaction and used genetic distance calculation algorithms to help determine the population structure for the studied loci. Our results showed a statistically different population structure distribution of both ACE I/D (P = 0.02, OR = 1.56, 95% CI = 1.05-2.33 for the D allele, white versus black subgroup) and angiotensinogen M/T polymorphism (P = 0.007, OR = 1.71, 95% CI = 1.14-2.58 for the T allele, white versus black subgroup). Different sample sizes are predicted to be determinant of the power to detect a given genotypic association with a particular phenotype when conducting two-locus association studies in admixtured populations. In addition, the postulated genetic model is also a major determinant of the power to detect any association in a given sample size. The present simulation study helped to demonstrate the complex interrelation among ethnicity, power of the association, and the postulated genetic model of action of a particular allele in the context of clustering studies. This information is essential for the correct planning and interpretation of future association studies conducted on this population.

Membrane proteins: functional and structural studies using reconstituted proteoliposomes and 2-D crystals

Reconstitution of membrane proteins into lipid bilayers is a powerful tool to analyze functional as well as structural areas of membrane protein research. First, the proper incorporation of a purified membrane protein into closed lipid vesicles, to produce proteoliposomes, allows the investigation of transport and/or catalytic properties of any membrane protein without interference by other membrane components. Second, the incorporation of a large amount of membrane proteins into lipid bilayers to grow crystals confined to two dimensions has recently opened a new way to solve their structure at high resolution using electron crystallography. However, reconstitution of membrane proteins into functional proteoliposomes or 2-D crystallization has been an empirical domain, which has been viewed for a long time more like "black magic" than science. Nevertheless, in the last ten years, important progress has been made in acquiring knowledge of lipid-protein-detergent interactions and has permitted to build upon a set of basic principles that has limited the empirical approach of reconstitution experiments. Reconstitution strategies have been improved and new strategies have been developed, facilitating the success rate of proteoliposome formation and 2-D crystallization. This review deals with the various strategies available to obtain proteoliposomes and 2-D crystals from detergent-solubilized proteins. It gives an overview of the methods that have been applied, which may be of help for reconstituting more proteins into lipid bilayers in a form suitable for functional studies at the molecular level and for high-resolution structural analysis.

Functional dyspepsia: relationship between clinical subgroups and Helicobacter pylori status in Western Turkey

The etiology of functional dyspepsia is not known. The objective of the present study was to determine the characteristics of functional dyspepsia in Western Turkey. We divided 900 patients with functional dyspepsia into three subgroups according to symptoms: ulcer-like (UL), 321 (35.6%), motility disorder-like (ML), 281 (31.2%), and the combination (C) of these symptoms, 298 (33.1%). All patients were submitted to endoscopic evaluation, with two biopsies taken from the cardia and corpus, and four from the antrum of the stomach. All biopsy samples were studied for Helicobacter pylori (Hp) density, chronic inflammation, activity, intestinal metaplasia, atrophy, and the presence of lymphoid aggregates by histological examination. One antral biopsy was used for the rapid urease test. Tissue cagA status was determined by PCR from an antral biopsy specimen by a random sampling method. We also determined the serum levels of tumor necrosis factor-alpha (TNF-alpha) and gastrin by the same method. Data were analyzed statistically by the Kolmogorov-Smirnov test and by analysis of variance. Hp and cagA positivity was significantly higher in the UL subgroup than in the others. The patients in the ML subgroup had the lowest Hp and cagA positivity and Hp density. The ML subgroup also showed the lowest level of Hp-induced inflammation among all subgroups. The serum levels of TNF-alpha and gastrin did not reveal any difference between groups. Our findings show a poor association of Hp with the ML subgroup of functional dyspepsia, but a stronger association with the UL and C subgroups.

Functional changes of human quadriceps muscle injured by eccentric exercise

The present study evaluated functional changes of quadriceps muscle after injury induced by eccentric exercise. Maximal isometric torque of quadriceps and the surface electromyography (root mean square, RMS, and median frequency, MDF) of the vastus medialis oblique (VMO) and vastus lateralis (VL) muscles were examined before, immediately after and during the first 7 days after injury. Serum creatine kinase (CK) levels and magnetic resonance imaging (MRI) were used to identify muscle injury. The subject was used as her own control and percent refers to pre-injury data. Experiments were carried out with a sedentary 23-year-old female. Injury was induced by 4 bouts of 15 maximal isokinetic eccentric contractions (angular velocity of 5º/s; range of motion from 40º to 110º of knee flexion). The isometric torque of the quadriceps (knee at 90º flexion) decreased 52% immediately after eccentric exercise and recovered on the 5th day. The highest reduction of RMS occurred on the 2nd day after injury in both VL (63%) and VMO (66%) and only VL recovered to the pre-injury level on the 7th day. Immediately after injury, the MDF decreased by 5 and 3% (VMO and VL, respectively) and recovered one day later. Serum CK levels increased by 109% on the 2nd day and were still increased by 32% on the 7th day. MRI showed large areas of injury especially in the deep region of quadriceps. In conclusion, eccentric exercise decreased the isometric torque and electromyographic signals of quadriceps muscle, which were recovered in one week, despite the muscle regeneration signals.

Electrophysiological evidence for glial-subtype glutamate transporter functional expression in rat cerebellar granule neurons

A glutamate-sensitive inward current (Iglu) is described in rat cerebellar granule neurons and related to a glutamate transport mechanism. We examined the features of Iglu using the patch-clamp technique. In steady-state conditions the Iglu measured 8.14 ± 1.9 pA. Iglu was identified as a voltage-dependent inward current showing a strong rectification at positive potentials. L-Glutamate activated the inward current in a dose-dependent manner, with a half-maximal effect at about 18 µM and a maximum increase of 51.2 ± 4.4%. The inward current was blocked by the presence of dihydrokainate (0.5 mM), shown by others to readily block the GLT1 isoform. We thus speculate that Iglu could be attributed to the presence of a native glutamate transporter in cerebellar granule neurons.

Functional role of a specific ganglioside in neuronal migration and neurite outgrowth

Cell migration occurs extensively during mammalian brain development and persists in a few regions in the adult brain. Defective migratory behavior of neurons is thought to be the underlying cause of several congenital disorders. Knowledge of the dynamics and molecular mechanisms of neuronal movement could expand our understanding of the normal development of the nervous system as well as help decipher the pathogenesis of neurological developmental disorders. In our studies we have identified and characterized a specific ganglioside (9-O-acetyl GD3) localized to the membrane of neurons and glial cells that is expressed in regions of cell migration and neurite outgrowth in the developing and adult rat nervous system. In the present article we review our findings that demonstrate the functional role of this molecule in neuronal motility.