Effects of Vaccination Against the H1N1 Virus on BDNF and TNF-α Plasma Levels in Pregnant Women.

BACKGROUND: Vaccination is a widespread strategy to protect women and their children during fetal development. However, there is a lack of knowledge about potential effects of H1N1 vaccination on concentration of cytokines that are important to mother's central nervous system functions and fetal neurodevelopment. OBJECTIVE: The main purpose of the present study was to evaluate such interaction. The specific goals were to study the effects of vaccination against the H1N1 virus on plasma levels of the Brain Derived Neurotrophic Factor(BDNF), Tumor Necrosis Factor-α (TNF-α) and TNF-α Receptors 1 and 2 (sTNFR1; sTNFR2), in different periods of gestation. METHODS: Data were obtained during the period of 6 months in 2010, from a sample of 94 pregnant women who were using the health care service of Conceição do Mato Dentro, a rural area in the state of Minas Gerais, Brazil. Seventeen women were in the first trimester of pregnancy, forty were in the second trimester and 37 were in the third trimester. Each of these groups was divided into two subgroups as follows: immunized against the H1N1 virus (I) and non-immunized (NI). Plasma concentrations of BDNF, TNF-α, sTNFR1 and sTNFR2 were measured using the sandwich ELISA. RESULTS: There was no difference in cytokine or neurotrophic factor levels evaluated between groups I and NI in any trimesters. CONCLUSION: These results show that the recommendation of vaccination against the H1N1 virus for all pregnant women as a public health measure could be considered safe, regarding aspects related to the role played by neurotrophin and cytokine, such as those of CNS development and immunological functions.

A New Member of Family 11 Polysaccharide Lyase, Rhamnogalacturonan Lyase (CtRGLf) from Clostridium thermocellum.

A thermostable, alkaline rhamnogalacturonan lyase (RG lyase) CtRGLf, of family 11 polysaccharide lyase from Clostridium thermocellum was cloned, expressed, purified and biochemically characterised. Both, the full-length CtRGLf (80 kDa) protein and its truncated derivative CtRGL (63.9 kDa) were expressed as soluble proteins and displayed maximum activity against rhamnogalacturonan I (RG I). CtRGLf showed maximum activity at 70 °C, while CtRGL at 60 °C. Both enzymes showed maximum activity at pH 8.5. CtRGLf and CtRGL do not show higher activity on substrates with high ß-D-galactopyranose (D-Galp) substitution, this catalytic property deviates from that of some earlier characterised RG lyases which prefer substrates with high D-Galp substitution. The enzyme activity of CtRGLf and CtRGL was enhanced by 1.5 and 1.3 fold, respectively, in the presence of 3 mM of Ca(2+) ions. The TLC analysis of the degraded products of RG I, released by the action of CtRGLf and CtRGL revealed the production of RG oligosaccharides as major products confirming their endolytic activity.

Role of pectinolytic enzymes identified in Clostridium thermocellum cellulosome.

The cloning, expression and characterization of three cellulosomal pectinolytic enzymes viz., two variants of PL1 (PL1A and PL1B) and PL9 from Clostridium thermocellum was carried out. The comparison of the primary sequences of PL1A, PL1B and PL9 revealed that these proteins displayed considerable sequence similarities with family 1 and 9 polysaccharide lyases, respectively. PL1A, PL1B and PL9 are the putative catalytic domains of protein sequence ABN54148.1 and ABN53381.1 respectively. These two protein sequences also contain putative carbohydrate binding module (CBM) and type-I dockerin. The associated putative CBM of PL1A showed strong homology with family 6 CBMs while those of PL1B and PL9 showed homology with family 35 CBMs. Recombinant derivatives of these three enzymes showed molecular masses of approximately 34 kDa, 40 kDa and 32 kDa for PL1A, PL1B and PL9, respectively. PL1A, PL1B and PL9 displayed high activity toward polygalacturonic acid and pectin (up to 55% methyl-esterified) from citrus fruits. However, PL1B showed relatively higher activity towards 55% and 85% methyl-esterified pectin (citrus). PL1A and PL9 showed higher activity on rhamnogalacturonan than PL1B. Both PL1A and PL9 displayed maximum activity at pH 8.5 with optimum temperature of 50°C and 60°C respectively. PL1B achieved highest activity at pH 9.8, under an optimum temperature of 50°C. PL1A, PL1B and PL9 all produced two or more unsaturated galacturonates from pectic substrates as displayed by TLC analysis confirming that they are endo-pectate lyase belonging to family 1 and 9, respectively. This report reveals that pectinolytic activity displayed by Clostridium thermocellum cellulosome is coordinated by a sub-set of at least three multi-modular enzymes.

Small angle X-ray scattering analysis of Clostridium thermocellum cellulosome N-terminal complexes reveals a highly dynamic structure.

Clostridium thermocellum produces the prototypical cellulosome, a large multienzyme complex that efficiently hydrolyzes plant cell wall polysaccharides into fermentable sugars. This ability has garnered great interest in its potential application in biofuel production. The core non-catalytic scaffoldin subunit, CipA, bears nine type I cohesin modules that interact with the type I dockerin modules of secreted hydrolytic enzymes and promotes catalytic synergy. Because the large size and flexibility of the cellulosome preclude structural determination by traditional means, the structural basis of this synergy remains unclear. Small angle x-ray scattering has been successfully applied to the study of flexible proteins. Here, we used small angle x-ray scattering to determine the solution structure and to analyze the conformational flexibility of two overlapping N-terminal cellulosomal scaffoldin fragments comprising two type I cohesin modules and the cellulose-specific carbohydrate-binding module from CipA in complex with Cel8A cellulases. The pair distribution functions, ab initio envelopes, and rigid body models generated for these two complexes reveal extended structures. These two N-terminal cellulosomal fragments are highly dynamic and display no preference for extended or compact conformations. Overall, our work reveals structural and dynamic features of the N terminus of the CipA scaffoldin that may aid in cellulosome substrate recognition and binding.

The connection between maternal thiamine shortcoming and offspring cognitive damage and poverty perpetuation in underprivileged communities across the world.

The acquisition of cognitive, sensory-motor and social emotional functions depend on a proper development of the Central Nervous System (CNS). This set of functions, known as intelligence, allows a better adaptation to the environment. In the last decades, an increase in the average of intelligence has been reported. However, such an increase cannot be observed in an equivalent way in economically and social underprivileged regions. Children from those regions are in great risk of being affected by mental retardation or impaired cognitive development. In later life they will, probably, be unable to transform and improve themselves and their communities, perpetuating the poverty of the region. Therefore, knowledge of factors involved in CNS development is a matter of health closely related to social improvement. Malnutrition throughout pregnancy and breastfeeding is clearly identifiable as a cause of damage in CNS development. Vitamin B1 (Thiamine) is a micronutrient important to the growth and maturity of the CNS. Thiamine shortcoming may affect 50% of pregnant women. Thiamine function in cerebral development is still not well known. There is a gap in the literature regarding systematical research about the blood thiamine concentration throughout the periods of gestation and breastfeeding. These studies are relevant in populations with a high level of nutritional vulnerability, because in a follow up offspring cognitive exam they could reveal if the maternal thiamine deficiency is related to child CNS impairment. This paper introduce the hypothesis that thiamine shortcoming during pregnancy and breastfeeding is directly related to cognitive impairment of child. Data about the neurophysiological role of thiamine, consequences of its shortcoming in experimental models, populations under the risk of thiamine shortcoming are presented. The hypothesis that maternal thiamine shortcoming causes damage related to child cognitive development needs to be considered. Thus, thiamine shortcoming during gestation and breastfeeding and its effects on children must be studied in many populations in order to know the magnitude of the problem and to indicate actions to overcome it.

Molecular architecture and structural transitions of a Clostridium thermocellum mini-cellulosome.

The cellulosome is a highly elaborate cell-bound multienzyme complex that efficiently orchestrates the deconstruction of cellulose and hemicellulose, two of the nature's most abundant polymers. Understanding the intricacy of these nanomachines evolved by anaerobic microbes could sustain the development of an effective process for the conversion of lignocellulosic biomass to bio-ethanol. In Clostridium thermocellum, cellulosome assembly is mediated by high-affinity protein:protein interactions (>10(9) M(-1)) between dockerin modules found in the catalytic subunits and cohesin modules located in a non-catalytic protein scaffold termed CipA. Whereas the atomic structures of several cellulosomal components have been elucidated, the structural organization of the complete cellulosome remains elusive. Here, we reveal that a large fragment of the cellulosome presents a mostly compact conformation in solution, by solving the three-dimensional structure of a C. thermocellum mini-cellulosome comprising three consecutive cohesin modules, each bound to one Cel8A cellulase, at 35 Å resolution by cryo-electron microscopy. Interestingly, the three cellulosomal catalytic domains are found alternately projected outward from the CipA scaffold in opposite directions, in an arrangement that could expand the area of the substrate accessible to the catalytic domains. In addition, the cellulosome can transit from this compact conformation to a multitude of diverse and flexible structures, where the linkers between cohesin modules are extended and flexible. Thus, structural transitions controlled by changes in the degree of flexibility of linkers connecting consecutive cohesin modules could regulate the efficiency of substrate recognition and hydrolysis.

Family 42 carbohydrate-binding modules display multiple arabinoxylan-binding interfaces presenting different ligand affinities.

Enzymes that degrade plant cell wall polysaccharides display a modular architecture comprising a catalytic domain bound to one or more non-catalytic carbohydrate-binding modules (CBMs). CBMs display considerable variation in primary structure and are grouped into 59 sequence-based families organized in the Carbohydrate-Active enZYme (CAZy) database. Here we report the crystal structure of CtCBM42A together with the biochemical characterization of two other members of family 42 CBMs from Clostridium thermocellum. CtCBM42A, CtCBM42B and CtCBM42C bind specifically to the arabinose side-chains of arabinoxylans and arabinan, suggesting that various cellulosomal components are targeted to these regions of the plant cell wall. The structure of CtCBM42A displays a beta-trefoil fold, which comprises 3 sub-domains designated as alpha, beta and gamma. Each one of the three sub-domains presents a putative carbohydrate-binding pocket where an aspartate residue located in a central position dominates ligand recognition. Intriguingly, the gamma sub-domain of CtCBM42A is pivotal for arabinoxylan binding, while the concerted action of beta and gamma sub-domains of CtCBM42B and CtCBM42C is apparently required for ligand sequestration. Thus, this work reveals that the binding mechanism of CBM42 members is in contrast with that of homologous CBM13s where recognition of complex polysaccharides results from the cooperative action of three protein sub-domains presenting similar affinities.

Low frequency of bipolar disorder, dopamine dysregulation syndrome, and punding in Brazilian patients with Parkinson's disease.

OBJECTIVE: To investigate the frequency of bipolar disorder, dopamine dysregulation syndrome and punding in Parkinson's disease patients from a Brazilian movement disorders clinic. METHOD: One hundred patients underwent a comprehensive psychiatric examination composed of MINI-plus and specific questionnaires to investigate dopamine dysregulation syndrome and punding. RESULTS: We identified, respectively, one and five Parkinson's disease patients with bipolar disorder type I and type II. All manic/hypomanic episodes occurred before Parkinson's disease onset. No patient was identified with dopamine dysregulation syndrome or punding. CONCLUSION: The frequency of manic/hypomanic episodes seems to decrease with Parkinson's disease onset, and local environmental factors (e.g. drug availability) may be responsible for the low frequency of dopamine dysregulation syndrome and punding in Brazilian Parkinson's disease patients.

Low frequency of bipolar disorder, dopamine dysregulation syndrome, and punding in Brazilian patients with Parkinson's disease / Baixa frequência de transtorno bipolar, síndrome de desregulação dopaminérgica e punding em pacientes brasileiros com doença de Parkinson

OBJECTIVE: To investigate the frequency of bipolar disorder, dopamine dysregulation syndrome and punding in Parkinson's disease patients from a Brazilian movement disorders clinic. METHOD: One hundred patients underwent a comprehensive psychiatric examination composed of MINI-plus and specific questionnaires to investigate dopamine dysregulation syndrome and punding. RESULTS: We identified, respectively, one and five Parkinson's disease patients with bipolar disorder type I and type II. All manic/hypomanic episodes occurred before Parkinson's disease onset. No patient was identified with dopamine dysregulation syndrome or punding. CONCLUSION: The frequency of manic/hypomanic episodes seems to decrease with Parkinson's disease onset, and local environmental factors (e.g. drug availability) may be responsible for the low frequency of dopamine dysregulation syndrome and punding in Brazilian Parkinson's disease patients.

OBJETIVO: Investigar a frequência de transtorno bipolar, síndrome de desregulação dopaminérgica e punding em pacientes com doença de Parkinson de uma clínica de movimentos anormais no Brasil. MÉTODO: Cem pacientes foram submetidos à avaliação psiquiátrica composta pelo MINI-Plus e questionários específicos para investigar síndrome de desregulação dopaminérgica e punding. RESULTADOS: Identificamos, respectivamente, um e cinco pacientes com transtorno bipolar tipo I e tipo II. Todos os episódios maníacos/hipomaníacos ocorreram antes do início da doença de Parkinson. Nenhum paciente foi identificado com síndrome de desregulação dopaminérgica ou punding. CONCLUSÃO: A frequência de episódios maníacos/ hipomaníacos parece declinar com o início da doença de Parkinson. Fatores ambientais locais (p.ex.: disponibilidade de drogas) podem ser responsáveis pela baixa frequência de síndrome de desregulação dopaminérgica e punding em pacientes brasileiros com doença de Parkinson.

Depression impairs executive functioning in Parkinson disease patients with low educational level.

OBJECTIVE: To assess overall cognitive performance and executive functioning of nondemented Parkinson disease (PD) patients, and the influence of variables such as depression and education on cognition. BACKGROUND: Cognitive dysfunction in PD is common even in early stages. Different variables have been identified as potential risk factors for cognitive decline in PD. Some of these variables, such as depression and educational level, are complexly interrelated. METHODS: Eighty-two (male:female 52:30) subjects underwent clinical assessment which included the Unified Parkinson's Disease Rating Scale, Schwab-England Scale, Hoehn-Yahr Scale, Beck Depression Inventory, the Frontal Assessment Battery (FAB), and the Mini-Mental State Examination (MMSE). RESULTS: Patients with higher education, younger age, and who had a younger age at disease onset performed better on both the FAB and MMSE. Severity of disease correlated with worse cognitive performance. Performance on the FAB, but not the MMSE, worsened with increased severity of depressive symptoms. When patients were divided into groups with lower (< or =4 y of schooling) and higher (> or =5 y of schooling) education, the FAB and Beck Depression Inventory correlated negatively only in the group with lower educational level. CONCLUSIONS: Patients with PD present with cognitive impairment even when nondemented. Depression may exacerbate executive dysfunction, especially in subjects with lower educational level.

Evidence for a dual binding mode of dockerin modules to cohesins.

The assembly of proteins that display complementary activities into macromolecular complexes is critical to cellular function. One such enzyme complex, of environmental significance, is the plant cell wall degrading apparatus of anaerobic bacteria, termed the cellulosome. The complex assembles through the interaction of enzyme-derived "type I dockerin" modules with the multiple "cohesin" modules of the scaffolding protein. Clostridium thermocellum type I dockerin modules contain a duplicated 22-residue sequence that comprises helix-1 and helix-3, respectively. The crystal structure of a C. thermocellum type I cohesin-dockerin complex showed that cohesin recognition was predominantly through helix-3 of the dockerin. The sequence duplication is reflected in near-perfect 2-fold structural symmetry, suggesting that both repeats could interact with cohesins by a common mechanism in wild-type (WT) proteins. Here, a helix-3 disrupted mutant dockerin is used to visualize the reverse binding in which the dockerin mutant is indeed rotated 180 degrees relative to the WT dockerin such that helix-1 now dominates recognition of its protein partner. The dual binding mode is predicted to impart significant plasticity into the orientation of the catalytic subunits within this supramolecular assembly, which reflects the challenges presented by the degradation of a heterogeneous, recalcitrant, insoluble substrate by a tethered macromolecular complex.

Galactomannan hydrolysis and mannose metabolism in Cellvibrio mixtus.

Galactomannan hydrolysis results from the concerted action of microbial endo-mannanases, manosidases and alpha-galactosidases and is a mechanism of intrinsic biological importance. Here we report the identification of a gene cluster in the aerobic soil bacterium Cellvibrio mixtus encoding enzymes involved in the degradation of this polymeric substrate. The family 27 alpha-galactosidase, termed CmAga27A, preferentially hydrolyse galactose containing polysaccharides. In addition, we have characterized an enzyme with epimerase activity, which might be responsible for the conversion of mannose into glucose. The role of the identified enzymes in the hydrolysis of galactomannan by aerobic bacteria is discussed.

The N-terminal family 22 carbohydrate-binding module of xylanase 10B of Clostridium themocellum is not a thermostabilizing domain.

Xylanase Xyn10B from Clostridium thermocellum is a modular enzyme that contains two family 22 carbohydrate binding modules N- (CBM22-1) and C- (CBM22-2) terminal of the family 10 glycoside hydrolase catalytic domain (GH10). In a previous study, we showed that removal of CBM22-1 reduces the resistance to thermoinactivation of the enzyme suggesting that this module is a thermostabilizing domain. Here, we show that it is the module border on the N-terminal side of GH10 that confers resistance to thermoinactivation and to proteolysis. Therefore, CBM22-1 does not function as a thermostabilizing domain and the role for this apparently non-functional CBM remains elusive.

Insights into the molecular determinants of substrate specificity in glycoside hydrolase family 5 revealed by the crystal structure and kinetics of Cellvibrio mixtus mannosidase 5A.

The enzymatic hydrolysis of the glycosidic bond is central to numerous biological processes. Glycoside hydrolases, which catalyze these reactions, are grouped into families based on primary sequence similarities. One of the largest glycoside hydrolase families is glycoside hydrolase family 5 (GH5), which contains primarily endo-acting enzymes that hydrolyze beta-mannans and beta-glucans. Here we report the cloning, characterization, and three-dimensional structure of the Cellvibrio mixtus GH5 beta-mannosidase (CmMan5A). This enzyme releases mannose from the nonreducing end of mannooligosaccharides and polysaccharides, an activity not previously observed in this enzyme family. CmMan5A contains a single glycone (-1) and two aglycone (+1 and +2) sugar-binding subsites. The -1 subsite displays absolute specificity for mannose, whereas the +1 subsite does not accommodate galactosyl side chains but will bind weakly to glucose. The +2 subsite is able to bind to decorated mannose residues. CmMan5A displays similar activity against crystalline and amorphous mannans, a property rarely attributed to glycoside hydrolases. The 1.5 A crystal structure reveals that CmMan5A adopts a (beta/alpha)(8) barrel fold, and superimposition with GH5 endo-mannanases shows that dramatic differences in the length of three loops modify the active center accessibility and thus modulate the specificity from endo to exo. The most striking and significant difference is the extended loop between strand beta8 and helix alpha8 comprising residues 378-412. This insertion forms a "double" steric barrier, formed by two short beta-strands that function to "block" the substrate binding cleft at the edge of the -1 subsite forming the "exo" active center topology of CmMan5A.

Cellulosome assembly revealed by the crystal structure of the cohesin-dockerin complex.

The utilization of organized supramolecular assemblies to exploit the synergistic interactions afforded by close proximity, both for enzymatic synthesis and for the degradation of recalcitrant substrates, is an emerging theme in cellular biology. Anaerobic bacteria harness a multiprotein complex, termed the "cellulosome," for efficient degradation of the plant cell wall. This megadalton catalytic machine organizes an enzymatic consortium on a multifaceted molecular scaffold whose "cohesin" domains interact with corresponding "dockerin" domains of the enzymes. Here we report the structure of the cohesin-dockerin complex from Clostridium thermocellum at 2.2-A resolution. The data show that the beta-sheet cohesin domain interacts predominantly with one of the helices of the dockerin. Whereas the structure of the cohesin remains essentially unchanged, the loop-helix-helix-loop-helix motif of the dockerin undergoes conformational change and ordering compared with its solution structure, although the classical 12-residue EF-hand coordination to two calcium ions is maintained. Significantly, internal sequence duplication within the dockerin is manifested in near-perfect internal twofold symmetry, suggesting that both "halves" of the dockerin may interact with cohesins in a similar manner, thus providing a higher level of structure to the cellulosome and possibly explaining the presence of "polycellulosomes." The structure provides an explanation for the lack of cross-species recognition between cohesin-dockerin pairs and thus provides a blueprint for the rational design, construction, and exploitation of these catalytic assemblies.