Facilitation of the main generator source of earthworm muscle contraction by a peripheral neuron

A constant facilitation of responses evoked in the earthworm muscle contraction generator neurons by responses evoked in the neurons of its peripheral nervous system was demonstrated. It is based on the proposal that these two responses are bifurcations of an afferent response evoked by the same peripheral mechanical stimulus but converging again on this central neuron. A single-peaked generator response without facilitation was demonstrated by sectioning the afferent route of the peripheral facilitatory modulatory response, or conditioning response (CR). The multipeaked response could be restored by restimulating the sectioned modulatory neuron with an intracellular substitutive conditioning stimulus (SCS). These multi-peaked responses were proposed to be the result of reverberating the original single peaked unconditioned response (UR) through a parallel (P) neuronal circuit which receives the facilitation of the peripheral modulatory neuron. This peripheral modulatory neuron was named "Peri-Kästchen" (PK) neuron because it has about 20 peripheral processes distributed on the surface of a Kästchen of longitudinal muscle cells on the body wall of this preparation as revealed by the Lucifer Yellow-CH-filling method.

Persistent attenuation and enhancement of the earthworm main muscle contraction generator response induced by repeated stimulation of a peripheral neuron

Responses evoked in the earthworm, Amynthas hawayanus, main muscle contraction generator M-2 (postsynaptic mechanical-stimulus-sensitive) neuron by threshold mechanical stimuli in 2-s intertrial intervals (ITI) were used as the control or unconditioned responses (UR). Their attenuation induced by decreasing these intervals in non-associative conditioning and their enhancement induced by associating the unconditioned stimuli (US) to a train of short (0.1 s) hyperpolarizing electrical substitutive conditioning stimuli (SCS) in the Peri-Kästchen (PK) neuron were measured in four parameters, i.e., peak numbers (N) and amplitude ()averaged from 120 responses, sum of these amplitudes (SAMP) and the highest peak amplitude (V) over a period of 4 min. Persistent attenuation similar to habituation was induced by decreasing the control ITI to 0.5 s and 2.0 s in non-associative conditioning within less than 4 min. Dishabituation was induced by randomly pairing one of these habituated US to an electrical stimulus in the PK neuron. All four parameters of the UR were enhanced by forward (SCS-US), but not backward (US-SCS), association of the US with 25, 100 and 250-Hz trains of SCS with 40-ms interstimulus intervals (ISI) for 4 min and persisted for another 4 min after turning off the SCS. The enhancement of these parameters was proportional to the SCS frequencies in the train. No UR was evoked by the SCS when the US was turned off after 4 min of classical conditioning.

Partial sequence and toxic effects of granulitoxin, a neurotoxic peptide from the sea anemone Bunodosoma granulifera

A neurotoxic peptide, granulitoxin (GRX), was isolated from the sea anemone Bunodosoma granulifera. The N-terminal amino acid sequence of GRX is AKTGILDSDGPTVAGNSLSGT and its molecular mass is 4958 Da by electrospray mass spectrometry. This sequence presents a partial degree of homology with other toxins from sea anemones such as Bunodosoma caissarum, Anthopleura fuscoviridis and Anemonia sulcata. However, important differences were found: the first six amino acids of the sequence are different, Arg-14 was replaced by Ala and no cysteine residues were present in the partial sequence, while two cysteine residues were present in the first 21 amino acids of other toxins described above. Purified GRX injected ip (800 µg/kg) into mice produced severe neurotoxic effects such as circular movements, aggressive behavior, dyspnea, tonic-clonic convulsion and death. The 2-h LD50 of GRX was 400 ± 83 µg/kg.

Effects of the partial removal of wood hemicelluloses on the properties of kraft pulp

The objective of this work was to study the effects of partial removal of wood hemicelluloses on the properties of kraft pulp.The work was conducted by extracting hemicelluloses (1) by a softwood chip pretreatment process prior to kraft pulping, (2) by alkaline extraction from bleached birch kraft pulp, and (3) by enzymatic treatment, xylanase treatment in particular, of bleached birch kraft pulp. The qualitative and quantitative changes in fibers and paper properties were evaluated. In addition, the applicability of the extraction concepts and hemicellulose-extracted birch kraft pulp as a raw material in papermaking was evaluated in a pilot-scale papermaking environment. The results showed that each examined hemicellulose extraction method has its characteristic effects on fiber properties, seen as differences in both the physical and chemical nature of the fibers. A prehydrolysis process prior to the kraft pulping process offered reductions in cooking time, bleaching chemical consumption and produced fibers with low hemicellulose content that are more susceptible to mechanically induced damages and dislocations. Softwood chip pretreatment for hemicellulose recovery prior to cooking, whether acidic or alkaline, had an impact on the physical properties of the non-refined and refined pulp. In addition, all the pretreated pulps exhibited slower beating response than the unhydrolyzed reference pulp. Both alkaline extraction and enzymatic (xylanase) treatment of bleached birch kraft pulp fibers indicated very selective hemicellulose removal, particularly xylan removal. Furthermore, these two hemicellulose-extracted birch kraft pulps were utilized in a pilot-scale papermaking environment in order to evaluate the upscalability of the extraction concepts. Investigations made using pilot paper machine trials revealed that some amount of alkalineextracted birch kraft pulp, with a 24.9% reduction in the total amount of xylan, could be used in the papermaking stock as a mixture with non-extracted pulp when producing 75 g/m2 paper. For xylanase-treated fibers there were no reductions in the mechanical properties of the 180 g/m2 paper produced compared to paper made from the control pulp, although there was a 14.2% reduction in the total amount of xylan in the xylanase-treated pulp compared to the control birch kraft pulp. This work emphasized the importance of the hemicellulose extraction method in providing new solutions to create functional fibers and in providing a valuable hemicellulose co-product stream. The hemicellulose removal concept therefore plays an important role in the integrated forest biorefinery scenario, where the target is to the co-production of hemicellulose-extracted pulp and hemicellulose-based chemicals or fuels.

Purification and partial characterization of Phaseolus vulgaris seed aminopeptidase

The aminopeptidase activity of Phaseolus vulgaris seeds was measured using L-Leu-p-nitroanilide and the L-aminoacyl-ß-naphthylamides of Leu, Ala, Arg and Met. A single peak of aminopeptidase activity on Leu-ß-naphthylamide was eluted at 750 µS after gradient elution chromatography on DEAE-cellulose of the supernatant of a crude seed extract. The effluent containing enzyme activity was applied to a Superdex 200 column and only one peak of aminopeptidase activity was obtained. SDS-polyacrylamide gel electrophoresis (10%) presented only one protein band with molecular mass of 31 kDa under reducing and nonreducing conditions. The aminopeptidase has an optimum pH of 7.0 for activity on all substrates tested and the highest Vmax/KM ratio for L-Leu-ß-naphthylamide. The enzyme activity was increased 40% by 0.15 M NaCl, inhibited 94% by 2.0 mM Zn2+, inhibited 91% by sodium p-hydroxymercuribenzoate and inhibited 45% by 0.7 mM o-phenanthroline and 30 µM EDTA. Mercaptoethanol (3.3 mM), dithioerythritol (1.7 mM), Ala, Arg, Leu and Met (70 µM), p-nitroaniline (0.25 mM) and ß-naphthylamine (0.53 mM) had no effect on enzyme activity when assayed with 0.56 mM of substrate. Bestatin (20 µM) inhibited 18% the enzyme activity. The aminopeptidase activity in the seeds decayed 50% after two months when stored at 4oC and room temperature. The enzyme is leucyl aminopeptidase metal- and thiol group-dependent.

Partial characterization of nif genes from the bacterium Azospirillum amazonense

Azospirillum amazonense revealed genomic organization patterns of the nitrogen fixation genes similar to those of the distantly related species A. brasilense. Our work suggests that A. brasilense nifHDK, nifENX, fixABC operons and nifA and glnB genes may be structurally homologous to the counterpart genes of A. amazonense. This is the first analysis revealing homology between A. brasilense nif genes and the A. amazonense genome. Sequence analysis of PCR amplification products revealed similarities between the amino acid sequences of the highly conserved nifD and glnB genes of A. amazonense and related genes of A. brasilense and other bacteria. However, the A. amazonense non-coding regions (the upstream activator sequence region and the region between the nifH and nifD genes) differed from related regions of A. brasilense even in nitrogenase structural genes which are highly conserved among diazotrophic bacteria. The feasibility of the 16S ribosomal RNA gene-based PCR system for specific detection of A. amazonense was shown. Our results indicate that the PCR primers for 16S rDNA defined in this article are highly specific to A. amazonense and can distinguish this species from A. brasilense.

Esophageal striated muscle contractions in patients with Chagas' disease and idiopathic achalasia

Chagas' disease causes degeneration and reduction of the number of intrinsic neurons of the esophageal myenteric plexus, with consequent absent or partial lower esophageal sphincter relaxation and loss of peristalsis in the esophageal body. The impairment of esophageal motility is seen mainly in the distal smooth muscle region. There is no study about esophageal striated muscle contractions in the disease. In 81 patients with heartburn (44 with esophagitis) taken as controls, 51 patients with Chagas' disease (21 with esophageal dilatation) and 18 patients with idiopathic achalasia (11 with esophageal dilatation) we studied the amplitude, duration and area under the curve of esophageal proximal contractions. Using the manometric method and a continuous perfusion system we measured the esophageal striated muscle contractions 2 to 3 cm below the upper esophageal sphincter after swallows of a 5-ml bolus of water. There was no significant difference in striated muscle contractions between patients with heartburn and esophagitis and patients with heartburn without esophagitis. There was also no significant difference between patients with heartburn younger or older than 50 years or between men and women or in esophageal striated muscle contractions between patients with heartburn and Chagas' disease. The esophageal proximal amplitude of contractions was lower in patients with idiopathic achalasia than in patients with heartburn. In patients with Chagas' disease there was no significant difference between patients with esophageal dilatation and patients with normal esophageal diameter. Esophageal striated muscle contractions in patients with Chagas' disease have the same amplitude and duration as seen in patients with heartburn. Patients with idiopathic achalasia have a lower amplitude of contraction than patients with heartburn.

Effect of hyperbaric oxygenation on the regeneration of the liver after partial hepatectomy in rats

The aim of the present study was to assess the influence of hyperbaric oxygenation (HBO) on rat liver regeneration before and after partial hepatectomy. Rats were sacrificed 54 h after 15% hepatectomy, liver and body weights were measured, and serum alanine transaminase (ALT) and aspartate transaminase (AST) activity and albumin levels were determined. The lipid peroxide level, as indicated by malondialdehyde production in the remnant liver was measured, and liver sections were analyzed by light microscopy. Five groups of 10 rats in each group were studied. The preHBO and pre-hyperbaric pressure (preHB) groups were treated before partial hepatectomy with 100% O2 and 21% O2, respectively, at 202,650 pascals, daily for 3 days (45 min/day). The control group was not treated before partial hepatectomy and recovered under normal ambient conditions after the procedure. Groups postHBO and postHB were treated after partial hepatectomy with HBO and HB, respectively, three times (45 min/day). The preHBO group presented a significant increase in the initiation of the regeneration process of the liver 54 h postoperatively. The liver/body weight ratio was 0.0618 ± 0.0084 in the preHBO compared to 0.0517 ± 0016 g/g in the control animals (P = 0.016). In addition, the preHBO group showed significant better liver function (evaluated by the lowest serum ALT and AST activities, P = 0.002 and P = 0.008, respectively) and showed a significant decrease in serum albumin levels compared to control (P < 0.001). Liver lipid peroxide concentration was lowest in the preHBO group (P < 0.001 vs control and postHBO group) and light microscopy revealed that the composition of liver lobules in the preHBO group was the closest to normal histological features. These results suggest that HBO pretreatment was beneficial for rat liver regeneration after partial hepatectomy.

Mutations in SRY and WT1 genes required for gonadal development are not responsible for XY partial gonadal dysgenesis

The WT1 transcription factor regulates SRY expression during the initial steps of the sex determination process in humans, activating a gene cascade leading to testis differentiation. In addition to causing Wilms' tumor, mutations in WT1 are often responsible for urogenital defects in men, while SRY mutations are mainly related to 46,XY pure gonadal dysgenesis. In order to evaluate their role in abnormal testicular organogenesis, we screened for SRY and WT1 gene mutations in 10 children with XY partial gonadal dysgenesis, 2 of whom with a history of Wilms' tumor. The open reading frame and 360 bp of the 5' flanking sequence of the SRY gene, and the ten exons and intron boundaries of the WT1 gene were amplified by PCR of genomic DNA. Single-strand conformation polymorphism was initially used for WT1 mutation screening. Since shifts in fragment migration were only observed for intron/exon 4, the ten WT1 exons from all patients were sequenced manually. No mutations were detected in the SRY 5' untranslated region or within SRY open-reading frame sequences. WT1 sequencing revealed one missense mutation (D396N) in the ninth exon of a patient who also had Wilms' tumor. In addition, two silent point mutations were found in the first exon including one described here for the first time. Some non-coding sequence variations were detected, representing one new (IVS4+85A>G) and two already described (-7ATG T>G, IVS9-49 T>C) single nucleotide polymorphisms. Therefore, mutations in two major genes required for gonadal development, SRY and WT1, are not responsible for XY partial gonadal dysgenesis.

Partial characterization and anticoagulant activity of a heterofucan from the brown seaweed Padina gymnospora

The brown algae Padina gymnospora contain different fucans. Powdered algae were submitted to proteolysis with the proteolytic enzyme maxataze. The first extract of the algae was constituted of polysaccharides contaminated with lipids, phenols, etc. Fractionation of the fucans with increasing concentrations of acetone produced fractions with different proportions of fucose, xylose, uronic acid, galactose, and sulfate. One of the fractions, precipitated with 50% acetone (v/v), contained an 18-kDa heterofucan (PF1), which was further purified by gel-permeation chromatography on Sephadex G-75 using 0.2 M acetic acid as eluent and characterized by agarose gel electrophoresis in 0.05 M 1,3 diaminopropane/acetate buffer at pH 9.0, methylation and nuclear magnetic resonance spectroscopy. Structural analysis indicates that this fucan has a central core consisting mainly of 3-ß-D-glucuronic acid 1-> or 4-ß-D-glucuronic acid 1 ->, substituted at C-2 with alpha-L-fucose or ß-D-xylose. Sulfate groups were only detected at C-3 of 4-alpha-L-fucose 1-> units. The anticoagulant activity of the PF1 (only 2.5-fold lesser than low molecular weight heparin) estimated by activated partial thromboplastin time was completely abolished upon desulfation by solvolysis in dimethyl sulfoxide, indicating that 3-O-sulfation at C-3 of 4-alpha-L-fucose 1-> units is responsible for the anticoagulant activity of the polymer.

CaMKIIdelta overexpression in hypertrophy and heart failure: cellular consequences for excitation-contraction coupling

Ca/calmodulin-dependent protein kinase IIdelta (CaMKIIdelta) is the predominant isoform in the heart. During excitation-contraction coupling (ECC) CaMKII phosphorylates several Ca-handling proteins including ryanodine receptors (RyR), phospholamban, and L-type Ca channels. CaMKII expression and activity have been shown to correlate positively with impaired ejection fraction in the myocardium of patients with heart failure and CaMKII has been proposed to be a possible compensatory mechanism to keep hearts from complete failure. However, in addition to these acute effects on ECC, CaMKII was shown to be involved in hypertrophic signaling, termed excitation-transcription coupling (ETC). Thus, animal models have shown that overexpression of nuclear isoform CaMKIIdeltaB can induce myocyte hypertrophy. Recent study from our laboratory has suggested that transgenic overexpression of the cytosolic isoform CaMKIIdeltaC in mice causes severe heart failure with altered intracellular Ca handling and protein expression leading to reduced sarcoplasmic reticulum (SR) Ca content. Interestingly, the frequency of diastolic spontaneous SR Ca release events (or opening of RyR) was greatly enhanced, demonstrating increased diastolic SR Ca leak. This was attributed to increased CaMKII-dependent RyR phosphorylation, resulting in increased and prolonged openings of RyR since Ca spark frequency could be reduced back to normal levels by CaMKII inhibition. This review focuses on acute and chronic effects of CaMKII in ECC and ETC. In summary, CaMKII overexpression can lead to heart failure and CaMKII-dependent RyR hyperphosphorylation seems to be a novel and important mechanism in ECC due to SR Ca leak which may be important in the pathogenesis of heart failure.

High expression of human carboxypeptidase M in Pichia pastoris: Purification and partial characterization

Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidyl-inositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. The present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. The cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. The cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. The recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. The enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein-1 min-1). This purification protocol permitted us to obtain 410 mg of the purified protein per liter of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa), suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes

Partial purification of tightly bound mitochondrial hexokinase from maize (Zea mays L.) root membranes

In mammals, hexokinase (HK) is strategically located at the outer membrane of mitochondria bound to the porin protein. The mitochondrial HK is a crucial modulator of apoptosis and reactive oxygen species generation. In plants, these properties related to HK are unknown. In order to better understand the physiological role of non-cytosolic hexokinase (NC-HK) in plants, we developed a purification strategy here described. Crude extract of 400 g of maize roots (230 mg protein) contained a specific activity of 0.042 µmol G6P min-1 mg PTN-1. After solubilization with detergent two fractions were obtained by DEAE column chromatography, NC-HK 1 (specific activity = 3.6 µmol G6P min-1 mg PTN-1 and protein recovered = 0.7 mg) and NC-HK 2. A major purification (yield = 500-fold) was obtained after passage of NC-HK 1 through the hydrophobic phenyl-Sepharose column. The total amount of protein and activity recovered were 0.04 and 18%, respectively. The NC-HK 1 binds to the hydrophobic phenyl-Sepharose matrix, as observed for rat brain HK. Mild chymotrypsin digestion did not affect adsorption of NC-HK 1 to the hydrophobic column as it does for rat HK I. In contrast to mammal mitochondrial HK, glucose-6-phosphate, clotrimazole or thiopental did not dissociate NC-HK from maize (Zea mays) or rice (Oryza sativa) mitochondrial membranes. These data show that the interaction between maize or rice NC-HK to mitochondria differs from that reported in mammals, where the mitochondrial enzyme can be displaced by modulators or pharmacological agents known to interfere with the enzyme binding properties with the mitochondrial porin protein.

Abnormal response of left ventricular systolic function to submaximal exercise in post-partial left ventriculotomy patients

Patients with heart failure who have undergone partial left ventriculotomy improve resting left ventricular systolic function, but have limited functional capacity. We studied systolic and diastolic left ventricular function at rest and during submaximal exercise in patients with previous partial left ventriculotomy and in patients with heart failure who had not been operated, matched for maximal and submaximal exercise capacity. Nine patients with heart failure previously submitted to partial left ventriculotomy were compared with 9 patients with heart failure who had not been operated. All patients performed a cardiopulmonary exercise test with measurement of peak oxygen uptake and anaerobic threshold. Radionuclide left ventriculography was performed to analyze ejection fraction and peak filling rate at rest and during exercise at the intensity corresponding to the anaerobic threshold. Groups presented similar exercise capacity evaluated by peak oxygen uptake and at anaerobic threshold. Maximal heart rate was lower in the partial ventriculotomy group compared to the heart failure group (119 ± 20 vs 149 ± 21 bpm; P < 0.05). Ejection fraction at rest was higher in the partial ventriculotomy group as compared to the heart failure group (41 ± 12 vs 32 ± 9%; P < 0.0125); however, ejection fraction increased from rest to anaerobic threshold only in the heart failure group (partial ventriculotomy = 44 ± 17%; P = non-significant vs rest; heart failure = 39 ± 11%; P < 0.0125 vs rest; P < 0.0125 vs change in the partial ventriculotomy group). Peak filling rate was similar at rest and increased similarly in both groups at the anaerobic threshold intensity (partial ventriculotomy = 2.28 ± 0.55 EDV/s; heart failure = 2.52 ± 1.07 EDV/s; P < 0.0125; P > 0.05 vs change in partial ventriculotomy group). The abnormal responses demonstrated here may contribute to the limited exercise capacity of patients with partial left ventriculotomy despite the improvement in resting left ventricular systolic function.

Glycyrrhizin attenuates endotoxin- induced acute liver injury after partial hepatectomy in rats

Massive hepatectomy associated with infection induces liver dysfunction, or even multiple organ failure and death. Glycyrrhizin has been shown to exhibit anti-oxidant and anti-inflammatory activities. The aim of the present study was to investigate whether glycyrrhizin could attenuate endotoxin-induced acute liver injury after partial hepatectomy. Male Wistar rats (6 to 8 weeks old, weighing 200-250 g) were randomly assigned to three groups of 24 rats each: sham, saline and glycyrrhizin. Rats were injected intravenously with lipopolysaccharide (LPS) 24 h after 70% hepatectomy. Glycyrrhizin, pre-administered three times with 24 h intervals 48 h before hepatectomy, prolonged the survival of rats submitted to partial hepatectomy and LPS injection, compared with saline controls. Glycyrrhizin was shown to attenuate histological hepatic changes and significantly reduced serum levels of aspartate aminotransferase, alanine aminotransferase, and lactic dehydrogenase, at all the indicated times (6 rats from each were sacrificed 1, 3, 6, and 9 h after LPS injection), compared with saline controls. Glycyrrhizin also significantly inhibited hepatocyte apoptosis by down-regulating the expression of caspase-3 and inhibiting the release of cytochrome C from mitochondria into the cytoplasm. The anti-inflammatory activity of glycyrrhizin may rely on the inhibition of release of tumor necrosis factor-a, myeloperoxidase activity, and translocation of nuclear factor-kappa B into the nuclei. Glycyrrhizin also up-regulated the expression of proliferating cell nuclear antigen, implying that it might be able to promote regeneration of livers harmed by LPS. In summary, glycyrrhizin may represent a potent drug protecting the liver against endotoxin-induced injury, especially after massive hepatectomy.