APGW-amide as an inhibitory neurotransmitter of Achatina fulica Ferussac

APGWamide (L-Ala-L-Pro-Gly-L-Trp-NH2) was purified from the ganglia of an African giant snail (Achatina fulica Ferussac). This peptide inhibited (hyperpolarized) more than half of the Achatina neurone types tested. This produced an outward current with the membrane conductance increase of RAPN (right anterior pallial neurone) under voltage clamp. The ED50 of the peptide was 6.2 x 10(-6) M (95% confidence limit: 5.0-7.8 x 10(-6) M) and the Emax was 3.9 +/- 0.2 nA. The effects were due to a membrane permeability increase to K+. The peptide is proposed as an inhibitory neurotransmitter of the Achatina neurones.     

Immunocytochemical identification of neuroendocrine markers in human cardiac paraganglion — like structures

Summary Paraganglion-like structures (PLS) containing chromaffin-positive cells have been reported to be present in the adult human heart. The present work was initiated in order to evaluate the densitity of these structures in the interatrial septum and to study the presence of immunoreactivity of their cells to NSE and PGP 9,5 antibodies, two neuroendocrine markers. Six hundred 6-m paraffin serial sections were obtained from the upper third of the interatrial septum from six adult human hearts. From 2 to 12 paraganglia were found in each case, and their principal cells stained positively with NSE and PGP 9,5 antibodies. Depending on how these PLS related to other cardiac structures, four different types were identified: Type I — True paraganglia (located adjacent to ganglia or nerve fibers); Type II — Free paraganglia (immersed in the interatrial adipose tissue, without evident connection to other structures); Type III — Intraganglionic paraganglia (located within the nervous ganglia); Type IV — Intramyocardic paraganglia (small nests of immunoreactive cells closely related to myocardiocyte bundles). These cardiac paraganglia, which probably belong to the visceral-autonomic group, may have a role in the regulation of the cardiac function and in the adaptive mechanisms of the heart. Its is also possible that they originate functioning and non-functioning tumours.Work supported by grants from FINEP and CNPq (Brazil)     

Recovery of alkaline proteases by membrane filtration

This work describes the recovery of an extracellular alkaline protease from fermentation broths of a Bacillus sp ATCC 21536, at pH=10.0 using ultrafiltration (MWCO 100,000) and microfiltration (0.1 m) membranes in hollow fiber devices. The influence of membrane pore size and polymeric material and membrane filtration performance was studied. High protein recoveries and high average flux rates were obtained with polysulfone membranes. A decrease of concentration polarization was obtained, simultaneously with enhancement of filtration flux rate and enzyme recovery by using submicron sized charged particles. These polymers lead to flocculation and adsorption of whole cells and soluble factors from the fermentation broth. The best results were obtaiend by combination of cationic (0.1%) and anionic (0.04%) polymers.     

Alveolar pressure measurement in open-chest rats.

In open-chest rats, alveolar pressure was measured with alveolar capsules connected via pliable tubing to inductive pressure transducers. By means of the interrupter technique during constant-flow inflation, it was possible to determine pulmonary static elastance (Est,L) and tissue and airway resistances (Rdiff,L and Rinit,L, respectively). In eight anesthetized paralyzed mechanically ventilated rats, 118 measurements of Rdiff,L and Est,L were performed over a wide range of flows and tidal volumes. There was excellent agreement between the data calculated using transpulmonary pressures and those computed using capsule pressures, the latter being measured at different points of the lung. In another group of rats studied under the same experimental conditions, two capsules were simultaneously placed on different pulmonary lobes. No regional differences in pulmonary mechanics could be detected in either experiment. In addition, alveolar pressure could also be measured accurately by a catheter inserted into lung parenchyma.     

Vagal function impairment after exercise training.

The present investigation was undertaken to evaluate the vagal function of trained (T) and sedentary (S) rats by use of different approaches in the same animal. After 13 wk of exercise training (treadmill for 1 h 5 times/wk at 26.8 m/min and 15% grade), T rats had a resting heart rate (HR) slightly but significantly lower than S rats (299 +/- 3 vs. 308 +/- 3 beats/min). T rats had marked reduction of the intrinsic HR (329 +/- 4 vs. 369 +/- 5 beats/min) after blockade by methylatropine and propranolol. They also exhibited depressed vagal and sympathetic tonus. Baroreflex bradycardia (phenylephrine injections) was reduced, bradycardic responses produced by electrical stimulation of the vagus were depressed, and responses to methacholine injection were decreased in T rats. Therefore several evidences of vagal function impairment were observed in T rats. The resting bradycardia after exercise training is more likely to be dependent on alterations of the pacemaker cells, inasmuch as the intrinsic HR was markedly reduced.     

Differential sensitivity of constitutive and facultative heterochromatin in orthopteran chromosomes to digestion by DNaseI

In situ pancreatic DNaseI digestions were used as probes to study the structural organization of facultative and constitutive heterochromatin during both mitotic and meiotic divisions. Three different types of heterochromatic regions from three insect species were chosen for this study. These regions had been previously characterized by in situ treatments with restriction endonucleases (AT and GC rich DNA sequences). Progressive increase in DNaseI concentration (from 10 to 200 ng/ml) or in incubation time (from 5 to 30 min) revealed a specific pattern of sequential digestion of the constitutive heterochromatic regions, the centromeric ones (AT-rich DNA) being the most resistant to DNaseI action. The interstitial C-bands (with AT or GC-rich DNA) were more sensitive to DNaseI, and the band 4.4 from Baetica ustalata was the most resistant of the non-centromeric bands. Similar results were obtained during meiosis, but increased accessibility to DNAseI was observed compared to mitosis. DNA methylation in the non-centromeric band 4.4 of B. ustulata could be responsible for its differential digestion with respect to the remaining intercalar heterochromatin. Facultatively heterochromatic regions (X chromosomes) were found to exhibit a differential response to DNaseI attack from mitosis to meiosis. While they behaved as cuchromatin during mitosis, they were the most resistant together with centromeric heterochromatin regions, during metaphase I and II. The different responses to digestion of the X chromosome and X-derived regions between somatic and meiotic divisions are probably a consequence of the changes in the organization of this chromosome during the facultative heterochromatinization process.     

Permeabilization of yeast for in situ determination of α-glucosidase

A quantitative in situ assay of yeast α-glucosidase involving permeabilization of the cells by freezing and thawing is described. The assay was applied to different strains in different physiological states and was shown to give results comparable to those obtained with total cell homogenates. The primary advantage of the in situ assay was the possibility of analyzing a large number of samples from the same culture during a growth curve using a very reduced cell mass.     

The plasma membrane of Saccharomyces cerevisiae. Isolation and some properties.

The isolation of Saccharomyces cerevisiae plasma membrane was carried out after hypotonic lysis of yeast protoplasts treated with concanavalin A by two independent methods: a, at low speed centrifugation and b, at high speed centrifugation in a density gradient. Several techniques (electron microscopic, enzymic, tagging, etc.) were used to ascertain the degree of purification of the plasma membranes obtained. The low speed centrifugation technique as compared with the other method gave a higher yield of plasma membranes with a similar degree of purification. Analysis of the yeast plasma membrane of normally growing cells by sodium dodecyl sulphate polyacrylamide gel electrophoresis showed at least 25 polypeptide bands. Twelve glycoprotein bands were also found, and their apparent molecular weights were determined. Treatment of the protoplasts with cycloheximide resulted in a significant decrease in the carbohydrate and protein content of the plasma membrane. The electrophoretic pattern of the plasma membrane of cycloheximide-treated cells showed a redistribution of the relative amounts of each protein band and a drastic reduction in the number of Schiff-positive bands. The isoelectric point of the most abundant proteins was low (pI 4) or lower than expected from previous data. A large part of the mannosyl transferase activity found in the cell (80%) was associated with the internal membranes, the remaining activity (20%) was located in the plasma membrane preparation. Part of the mannosyl transferase activity of the cells is located at the plasma membrane surface. Invertase (an external mannoprotein) is found in both the plasma and internal membranes, and as the specific activity dropped significantly following cycloheximide treatment of the cells, it is suggested that these membranes systems are the structures for the glycosylation of a precursor invertase and its subsequent release into the periplasmic space. Other transferase found in the plasma membrane preparation transfers glucose residues from UDPglucose to a poly(alpha(1 leads to 4) polymer identified as glycogen.