Effects of anodal transcranial direct current stimulation over motor cortex on resting-state brain activity in the early subacute stroke phase: A power spectral density analysis.

INTRODUCTION: Despite promising results, the effects of transcranial direct current stimulation (tDCS) in the early stages of stroke and its impact on brain activity have been poorly studied. Therefore, this study aimed to investigate the effect of tDCS applied over the ipsilesional motor cortex on resting-state brain activity in the early subacute phase of stroke. METHODS: This is a pilot, randomized, double-blind, proof-of-concept study. The patients with stroke were randomly assigned into two groups: anodal tDCS (A-tDCS) or sham tDCS (S-tDCS). For A-tDCS, the anode was placed over the ipsilesional motor cortex, while the cathode was placed over the left or right supraorbital area (Fp2 for left stroke or Fp1 for right stroke). For the real stimulation, a constant current of 1.0 mA was delivered for 20 min and then ramped down linearly for 30 s, maintaining a resistance below 10 kΩ. For the sham stimulation, the stimulator was turned on, and the current intensity was gradually increased for 30 s, tapered off over 30 s, and maintained for 30 min without stimulation. Each stimulation was performed for three consecutive sessions with an interval of 1 h between them. The primary outcome was spectral electroencephalography (EEG) analysis based on the Power Spectral Density (PSD) determined by EEG records of areas F3, F4, C3, C4, P3, and P4. Brain Vision Analyzer software processed the signals, EEG power spectral density (PSD) was calculated before and after stimulation, and alpha, beta, delta, and theta power were analyzed. The secondary outcomes included hemodynamic variables based on the difference between baseline (D0) and post-intervention session (D1) values of systolic (SBP) and diastolic (DBP) blood pressure, heart rate (HR), respiratory rate (RR) and peripheral oxygen saturation (SPO2). Mann-Whitney test was used to compare position measurements of two independent samples; Fisher's exact test was used to compare two proportions; paired Wilcoxon signed-rank test was used to compare the median differences in the within-group comparison, and Spearman correlations matrix among spectral power analysis between EEG bands was performed to verify consistency of occurrence of oscillations. Statistical significance was set at P < 0.05. RESULTS: An increase in PSD in the alpha frequency in the P4 region was observed after the intervention in the A-tDCS group, as compared to the placebo group (before = 6.13; after = 10.45; p < 0.05). In the beta frequency, an increase in PSD was observed in P4 (before = 4.40; after = 6.79; p < 0.05) and C4 (before = 4.43; after = 6.94; p < 0.05) after intervention in the A-tDCS group. There was a reduction in PSD at delta frequency in C3 (before = 293.8; after = 58.6; p < 0.05) after intervention in the A-tDCS group. In addition, it was observed a strong relationship between alpha and theta power in the A-tDCS group before and after intervention. However, the sham group showed correlations between more power bands (alpha and theta, alpha and delta, and delta and theta) after intervention. There was no difference in hemodynamic variables between the intra- (before and after stimulation) and inter-groups (mean difference). CONCLUSION: Anodal tDCS over the ipsilesional motor cortex had significant effects on the brain electrical activity in the early subacute stroke phase, increasing alpha and beta wave activities in sensorimotor regions while reducing slow delta wave activity in motor regions. These findings highlight the potential of anodal tDCS as a therapeutic intervention in the early stroke phase.

Description and management of cutaneous side effects during erlotinib and cetuximab treatment in lung and colorectal cancer patients: a prospective and descriptive study of 19 patients.

Erlotinib and cetuximab are human epidermal growth factor receptor inhibitors (EGFRI) that are approved in monotherapy for the treatment of patients with locally advanced or metastatic non-small cell lung cancer after failure of at least one prior chemotherapy regimen. Papulopustular eruptions are the most frequent adverse effect, their occurrence being associated with increased survival in some studies. We describe 19 patients who presented with a rash located mainly to the face and trunk, without presence of comedones, shortly after initiation of EGFRI therapy. We present our algorithm to manage these patients and their respective responses. We also report other therapeutic options and cutaneous alterations that may be seen.

Novel insights in the use of hydrolytic enzymes secreted by fungi with biotechnological potential.

Entomopathogenic and mycoparasitic fungi synthesize hydrolytic enzymes such as chitinases, proteinases and beta-glucanases. These enzymes can act synergistically, helping fungi to control insect pests and pathogens that attack productive crops, and offer potential economic benefit to agribusiness. A number of hydrolytic enzymes have also been utilized in industrial applications. This review focuses on biochemical and structural analyses of fungal enzymes, together with current research information on secretion mechanisms.

An antifungal peptide from passion fruit (Passiflora edulis) seeds with similarities to 2S albumin proteins.

An actual worldwide problem consists of an expressive increase of economic losses and health problems caused by fungi. In order to solve this problem, several studies have been concentrating on the screening of novel plant defence peptides with antifungal activities. These peptides are commonly characterized by having low molecular masses and cationic charges. This present work reports on the purification and characterization of a novel plant peptide of 5.0 kDa, Pe-AFP1, purified from the seeds of passion fruit (Passiflora edulis). Purification was achieved using a Red-Sepharose Cl-6B affinity column followed by reversed-phase chromatography on Vydac C18-TP column. In vitro assays indicated that Pe-AFP1 was able of inhibiting the development of the filamentous fungi Trichoderma harzianum, Fusarium oxysporum, and Aspergillus fumigatus with IC50 values of 32, 34, and 40 microg ml(-1), respectively, but not of Rhyzoctonia solani, Paracoccidioides brasiliensis and Candida albicans. This protein was also subjected to automated N-terminal amino acid sequence, showing high degree of similarities to storage 2S albumins, adding a new member to this protein-defence family. The discovery of Pe-AFP1 could contribute, in a near future, to the development of biotechnological products as antifungal drugs and transgenic plants with enhanced resistance to pathogenic fungi.

Increased expression of Bcl-xL and c-Myc is associated with transformation by Abelson murine leukemia virus.

Transformation mediated by the v-Abl oncoprotein, a tyrosine kinase encoded by the Abelson murine leukemia virus, is a multi-step process requiring genetic alterations in addition to expression of v-Abl. Loss of p53 or p19ARF was previously shown to be required for Abelson murine leukemia virus transformation of primary mouse embryonic fibroblasts (MEFs). By comparing gene expression patterns in primary p53-/- MEFs acutely infected with the v-Abl retrovirus, v-Abl-transformed MEF clones, and v-Abl-transformed MEF clones treated with Abl kinase inhibitor STI 571, we have identified additional genetic alterations associated with v-Abl transformation. Bcl-xL mRNA was elevated in three of five v-Abl-transformed MEF clones. In addition, elevated expression of c-Myc mRNA, caused either by c-myc gene amplification or by enhanced signaling via STAT3, was observed in five v-Abl-transformed MEF clones. The data suggest that increases in cell survival associated with Bcl-xL and increases in cell growth associated with c-Myc facilitate the transformation process dependent on constitutive mitogenic signaling by v-Abl.

Effects of dopaminergic and cholinergic interactions on rat behavior.

The present work studied the effects of dopaminergic and muscarinic receptor agonists and antagonists on rat locomotor activity and catalepsy. Results showed that carbachol at the highest dose used (10 mg/kg, p.o.) decreased and pimozide at the dose used abolished locomotor activity. Atropine at a low dose (1 mg/kg, p.o.) increased and at a high dose decreased this parameter. Mazindol at a high dose also increased locomotor activity. A significant and dose-dependent increase in the time on the bar was observed in animals treated with carbachol or pimozide as compared to controls. The increase observed with pimozide was greater than 60 s. Effects of carbachol on locomotor activity were observed already after the first drug exposure, but the increased time on bar produced by this drug in the test of catalepsy was observed only after repeated exposure (7th day). The effect of the highest dose (10 mg/kg, p.o.) of atropine (decreased activity) as related to the lowest one was evident at the 7th day, but the increased locomotor activity seen at the low dose was detected already at the first day. There was a predominance of the effect of pimozide on the open field as well as on catalepsy after its association with each one of the three doses of carbachol. The association of atropine and mazindol did not seem to alter locomotor activity and catalepsy as related to each drug alone. Our results indicate that interactions between dopaminergic and cholinergic systems play an important role on behavior and motor functions.

Structural relatedness of distinct determinants recognized by monoclonal antibody TP25.99 on beta 2-microglobulin-associated and beta 2-microglobulin-free HLA class I heavy chains.

The association of HLA class I heavy chains with beta2-microglobulin (beta2m) changes their antigenic profile. As a result, Abs react with either beta2m-free or beta2m-associated HLA class I heavy chains. An exception to this rule is the mAb TP25.99, which reacts with both beta2m-associated and beta2m-free HLA class I heavy chains. The reactivity with beta2m-associated HLA class I heavy chains is mediated by a conformational determinant expressed on all HLA-A, -B, and -C Ags. This determinant has been mapped to amino acid residues 194-198 in the alpha3 domain. The reactivity with beta2m-free HLA class I heavy chains is mediated by a linear determinant expressed on all HLA-B Ags except the HLA-B73 allospecificity and on <50% of HLA-A allospecificities. The latter determinant has been mapped to amino acid residues 239-242, 245, and 246 in the alpha3 domain. The conformational and the linear determinants share several structural features, but have no homology in their amino acid sequence. mAb TP25.99 represents the first example of a mAb recognizing two distinct and spatially distant determinants on a protein. The structural homology of a linear and a conformational determinant on an antigenic entity provides a molecular mechanism for the sharing of specificity by B and TCRs.

[Clinical study of falciparum malaria in children in Manaus, AM, Brazil]. / Estudo clínico da malária falciparum em crianças em Manaus, AM, Brasil.

The clinical characteristics of falciparum malaria were studied among 61 children, aged 0 to 14 treated at a reference center in Manaus, from October to December 1997. The symptoms observed were fever (98.4%), headache (80.3%), chills (68.9%), perspiration (65. 6%), myalgia (59.0%), nausea (54.1%), lumbar pain (49.2%), vomiting (49.2%), cough (45.9%), arthralgia (31.1%), diarrhea (34.4%), dyspnea (8.2%), convulsions (8.2%) and dizziness (4.9%). Pallor and anaemia were found more frequently in children under five years old. Anaemia was associated with high levels of parasitaemia. Fifty-eight (91.5%) patients had uncomplicated malaria, 3 (4.9%) had severe malaria and the lethality was 1.6%.

[RIII mefloquine resistance in children with falciparum malaria in Manaus, AM, Brazil]. / Resistência à mefloquina do tipo RIII em crianças com malária falciparum em Manaus, AM, Brasil.

We report the occurrence of resistance to mefloquine 20mg/day in 51 children with falciparum malaria treated, at reference center of Manaus, Brazil, from October to December 1997. All children were evaluated at day 3, 5, 7, 14, 21, 28 and 35 of treatment. Clinical and parasitological cure criteria were adopted. The incidence of RIII mefloquine resistance was 5.9% (IC 95% 1.5-17.2). The cure/resistance proportion was 20:1 and cure/severity was 62:1. These findings suggest the importance of mefloquine resistance within this group of children.

Regulation of 36-kDa beta-1,3-glucanase synthesis in Trichoderma harzianum.

The effect of carbon sources on the level of beta-1,3-glucanases in the culture filtrates of Trichoderma harzianum (Tc) was investigated. Enzyme activity was detected in all carbon sources, but highest levels were found when laminarin and purified cell walls were used. Three isoforms of beta-1,3-glucanase were produced during growth of the fungus on purified cell walls. Two isoforms were produced on chitin, chitosan, N-acetylglucosamine and laminarin, while only one was detected when the fungus was grown on cellulose and glucose. A 36-kDa beta-1,3-glucanase (GLU36) was secreted from T. harzianum (Tc) grown on all carbon sources tested as demonstrated by Western blot analysis. We found that a significant increase in the level of GLU36 in the culture filtrate follows glucose exhaustion, suggesting that this enzyme is controlled by carbon catabolite repression.

Analysis by NMR spectroscopy of the structural homology between the linear and the cyclic peptide recognized by anti-human leukocyte antigen class I monoclonal antibody TP25.99*.

The anti-human leukocyte antigen (HLA) class I monoclonal antibody (mAb) TP25.99 has a unique specificity since it recognizes both a conformational and a linear determinant expressed on the beta(2)-mu-associated and beta(2)-mu-free HLA class I heavy chains, respectively. Previously, we reported the identification of a cyclic and a linear peptide that inhibits mAb TP25.99 binding to the beta(2)-mu-associated and beta(2)-mu-free HLA class I heavy chains (S. A. Desai, X. Wang, E. J. Noronha, Q. Zhou, V. Rebmann, H. Grosse-Wilde, F. J. Moy, R. Powers, and S. Ferrone, submitted for publication). The linear X(19) and cyclic LX-8 peptides contain sequence homologous to residues 239-242, 245, and 246 and to residues 194-198, respectively, of HLA class I heavy chain alpha(3) domain. Analysis by two-dimensional transfer nuclear Overhauser effect spectroscopy of the induced solution structures of the linear X(19) and cyclic LX-8 peptides in the presence of mAb TP25.99 showed that the two peptides adopt a similar structural motif despite the lack of sequence homology. The backbone fold is suggestive of a short helical segment followed by a tight turn, reminiscent of the determinant loop region (residues 194-198) on beta(2)-mu-associated HLA class I heavy chains. The structural similarity between the linear X(19) and cyclic LX-8 peptides and the lack of sequence homology suggests that mAb TP25.99 predominantly recognizes a structural motif instead of a consensus sequence.

Characterization of a 29-kDa beta-1,3-glucanase from Trichoderma harzianum.

A beta-1,3-glucanase, from culture filtrates of Trichoderma harzianum, was purified in sequential steps by gel filtration, hydrophobic interaction and ion exchange chromatography. A typical procedure provided 69-fold purification with 0.32% yield. The molecular mass of the protein was found to be approximately 29 kDa, as estimated by SDS-PAGE on a 10% slab gel. The K(M) and V(max) values for beta-1,3-glucanase, using laminarin as substrate, were 1. 72 mg ml(-1) and 3.10 U ml(-1), respectively. The pH optimum for the enzyme was pH 4.4 and maximum activity was obtained at 50 degrees C. The enzyme was strongly inhibited by HgCl(2) and SDS. These results suggest that each beta-1,3-glucanase produced by T. harzianum is different and is probably encoded by different genes.

The epidemiology of malaria in Rondonia (Western Amazon region, Brazil): study of a riverine population.

We report on a longitudinal study concerning the incidence of malaria in a riverine population (Portuchuelo) settled on the riverbanks of Rio Madeira, in the State of Rondonia, Brazil. We found the incidence of malaria to be seasonal, prevailing in the dry months of June and July. The Annual Parasite Index (API) was 292/1000 inhabitants, almost three times that of the state of Rondonia for the same period. In contrast with other studied Rondonian populations, malaria in Portuchuelo was more prevalent in youngsters < 16 years old, particularly in the 0-1 year age group. Adults were relatively spared, particularly those over 50 years. Besides being indicative of indoor transmission, these facts may suggest the existence of a certain degree of acquired resistance to infection and/or of lessened symptoms in older people. Riverine populations are spread over the entire Amazon region where most of its members were born. Due to the permanent presence of malaria among riverine populations, we are proposing that they may act as perennial reserves of malaria and, therefore, as sources of infection for migrants or eventual settlers at their vicinity. To date, the opposite view has been generally held. Anopheles darlingi, the main vector species in the area, is essentially sylvatic, which contributes to make the control of malaria highly problematic. The only hopes for control rest on permanent surveillance and the prompt treatment of patients, which are also problematic considering the vastness of the Amazon region and the remoteness of some of its riverine settlements.

Limited diversity of human scFv fragments isolated by panning a synthetic phage-display scFv library with cultured human melanoma cells.

To broaden the specificity of the Abs recognizing human melanoma-associated Ags (MAAs), we have isolated human single-chain fragment of the V region (scFv) fragments by panning the synthetic phage Ab library (#1) with the human melanoma cell lines S5 and SK-MEL-28. All of the isolated scFv fragments reacted with the mouse mAb defined high molecular weight melanoma-associated Ag (HMW-MAA). scFv #70 immunoprecipitates the two characteristic subunits of HMW-MAA, while scFv #28 only immunoprecipitates its large subunit. These results challenge the current view regarding the structure of HMW-MAA and indicate that it consists of two independent subunits. The human scFv fragments share some similarities with the mouse anti-HMW-MAA mAb. Like mAb 149.53 and 225.28, scFv #28 reacts with rat B49 neural cells that express a homologue of HMW-MAA. scFv #70 reacts with a determinant that is spatially close to the one identified by mAbs 149.53, VT68.2, and VT86. Besides suggesting similarities in the recognition of human melanoma cells by the mouse and human Ab repertoire, these results indicate that the Abs isolated from synthetic Ab libraries resemble those that are found in natural Ab repertoires. The restricted diversity of the scFv fragments that were isolated by panning synthetic Ab libraries with different melanoma cell lines suggests that certain Ags, like HMW-MAA, are immunodominant in vitro. This phenomenon, which parallels the in vivo immunodominance of certain Ags, implies that the antigenic profile of the cells used for panning determines the specificity of the preponderant population of isolated Abs.

Characterization of human anti-high molecular weight-melanoma-associated antigen single-chain Fv fragments isolated from a phage display antibody library.

The human high molecular weight-melanoma-associated antigen (HMW-MAA) meets the criteria to be used as an immunogen for immunotherapy of malignant melanoma, because it is expressed by a large percentage of melanoma lesions with limited heterogeneity and has a restricted distribution in normal tissues. The high immunogenicity of the HMW-MAA in BALB/c mice has resulted in the development of a large number of anti-HMW-MAA monoclonal antibodies (mAbs). In contrast, no human anti-HMW-MAA mAbs have been described. Because the latter may serve as useful probes to characterize the antigenic profile of the HMW-MAA, human anti-HMW-MAA single-chain fragments of the variable region (scFvs) were isolated by panning synthetic scFv library 1 on purified HMW-MAA. Colony hybridization studies and nucleotide sequence analysis revealed that scFv 19, 44, 56, and 61 belong to the V(H)3 gene family and use the DP-38 germ-line gene segment but have a diverse third complementarity-determining region. The human scFvs share some characteristics with mouse anti-HMW-MAA mAb but also display some distinct features. Like mouse mAbs, human scFvs recognize determinants of HMW-MAA with a heterogeneous cellular and molecular distribution in human melanoma cells. Furthermore, like some mouse mAbs, human scFvs react with rat neural cells expressing the chondroitin sulfate proteoglycan NG2, which shows 81% homology to the HMW-MAA. However, at variance with mouse mAbs, the human scFvs show poor reactivity with guinea pig melanoma cells. Lastly, human scFv 61 stains both benign and malignant lesions of melanocytic origin, although with a lower frequency than mouse mAbs. Analysis of the clinical significance of the differential expression of the scFv 61-defined determinant in melanoma lesions will be facilitated by its reactivity with formalin-fixed melanoma lesions. In contrast to mouse mAb, scFv 61 immunoprecipitates the >450-kDa chondroitin sulfate proteoglycan component of the HMW-MAA, but not its 250-kDa subunit from melanoma cells. Thus, contrary to the current view about the structure of HMW-MAA, our results demonstrate that the two components are not associated. The described scFv antibodies, which represent the first example of human anti-HMW-MAA antibodies, have provided novel information about the structure of this antigen. Future studies will assess the impact of these in vitro-assembled antibody fragments on the identification of antigenic determinants of the HMW-MAA that can be recognized by the human immune system.

Excretion and distribution of ammonia and the influence of boundary layer acidification in embryonic rainbow trout (Oncorhynchus mykiss)

This study examined ammonia excretion by embryos of the rainbow trout (Oncorhynchus mykiss). The distribution of ammonia in relation to the H+ distribution and electrical potential was determined. The influence of the pH of the unstirred layer (USL) of water next to the external surface of the embryo was also assessed. Eyed-up embryos (35­40 days post-fertilization) were exposed to various external water conditions [pH 6.0, pH 10.0, 1.6 mmol l-1 NaCl, 0.0 mmol l-1 NaCl, 0.2 mmol l-1 NH4Cl, 2.5 mmol l-1 borax buffer (Na2B4O7.10H2O), 2.5 mmol l-1 Hepes, 0.1 mmol l-1 amiloride] for 30 min and ammonia excretion rates, ammonia concentration in the perivitelline fluid (PVF) and yolk, and the pH of the PVF, yolk and USL were measured. The rate of ammonia excretion was dependent, in part, on the partial pressure gradient of NH3 ( PNH3) from the PVF to the USL. Exposure to water of pH 6 increased, whereas NH4Cl or pH 10 exposure decreased, ammonia excretion rates. Elevated external Na+ levels also influenced the rate of ammonia excretion, but neither Na+-free water nor amiloride had any effect. The distribution of ammonia between the PVF and USL was dependent on the H+ distribution, but ammonia was distributed according to the electrical potential between the PVF and yolk. The USL was 0.32 pH units more acidic than the bulk water. Addition of buffer to the external water eliminated the acid USL and decreased ammonia excretion rates. We conclude that rainbow trout embryos excrete ammonia primarily as NH3, but when external Na+ levels are elevated, ammonia excretion may be independent of the PNH3 gradient. The acidic USL next to the chorion probably facilitates NH3 diffusion by maintaining the PNH3 through the conversion of NH3 to NH4+ upon entry into the USL.