Quantitative aspects of cytochemical methods for acetylcholinesterase studied with a cytochemical model system

Summary A model system of polyacrylamide films containing the Triton extract of rat brain homogenate was applied to investigate quantitatively some aspects of three methods for the cytochemical demonstration of acetylcholinesterase activity (Lewis 1961; Karnovsky and Roots 1964; Tsuji 1974).Biochemical determinations showed that about 90% of the acetylcholinesterase activity originally present in the Triton extract were still detectable in the films. The relationship of the formation of cuprous thiocholine iodide in the case of the methods of Lewis (1961) or Tsuji (1974) and of cupric ferrocyanide at the reaction of Karnovsky and Roots (1964) to either enzyme concentration or incubation time were tested in detail. The results showed that for the method of Tsuji and, with some restrictions, also for the method of Karnovsky and Roots a linearity exists in these two respects. In the case of the Lewis technique, an approximate linearity between the amount of reaction product and incubation time could only be found from 90 min onward, but no linearity was detected in relation to the enzyme concentration. At low enzyme concentrations, too little white precipitate was formed in comparison to higher ones. Therefore it is suggested that this technique, as compared to the methods of Tsuji and Karnovsky and Roots, probably is less suitable as a quantitative cytochemical method.This word was performed while one of us (Dr. Andrä) was in receipt of a visitor grant from the Netherlands Organization for the Advancement of Pure Research (ZWO)     

Estimation of the mechanical parameters of the human respiratory system

A numerical algorithm has been developed for the estimation of the mechanical parameters of the human respiratory system. In order to estimate the pulmonary resistance and the dynamic pulmonary elastance, the transpulmonary pressure and the airflow at the mouth or nose are expanded in Chebyshev series. The nonlinear mathematical lung model and a set of measurements for airflow and pressure are then handled by the numerical technique. The lung model includes a component to account for turbulent flow in the larynx and trachea. This contribution presents an alternative method for lung parameter estimation and differs from most existing methods in that it does not need measurements for the tidal volume. It therefore eliminates the use of a body plethysmograph. The method may also find potential application to various other parameter identification problems.     

Laryngeal papillomas: local cellular immune response,keratinization and viral antigen

Virchows Archiv B Cell Pathology - Various parameters of the local cellular response have been studied in 16 laryngeal papillomas from ten patients with recurrent papillomas as well as normal...     

Sodium channel activity in brain membrane fractions isolated from rats of different ages

The Na⁺ channel activity (tetrodotoxin sensitive ²²Na⁺ flux induced by veratridine and/or anemone toxin II) was studied in two fractions of brain cell plasma membranes, named A and B, isolated by the method of Gray and Whittaker ((1962) J. Anat. 96, 79–87) from rats 5, 10, 30 and 60 days old. The ²²Na⁺ flux was measured in membrane vesicles formed by the isolated membranes, in the absence of drugs (control), in the presence of veratridine, and in the presence of veratridine plus tetrodotoxin. Fraction A consists primarily of neuronal and glial membranes in rats of 5 and 10 days of age, while in the older rats this fraction becomes enriched in myelin. In Fraction A of 5-day-old and 10-day-old rats, veratridine (25 μM) increases the ²²Na⁺ flux 2.4- and 1.6-fold, respectively, and the increment continues to diminish with age, until it becomes negligible in the 60-day-old rats. Fraction B consists of synaptosomes and membrane vesicles, and at the four ages studied veratridine (25 μM) causes an increment of the ²²Na⁺ flux of about 2.5-fold. Fractions A and B from 10-day-old rats, and Fraction B from 60-day-old rats, which are sensitive to veratridine, also respond to anemone toxin II. When veratridine is used in presence of anemone toxin II (0.5 μM), the $\text{K}{}_{0.5}$ for veratridine is diminished and the maximum ²²Na⁺ flux is increased. The increments of ²²Na⁺ flux caused by veratridine and/or anemone toxin II in Fractions A and B are blocked by tetrodotoxin ($\text{K}{}_{0.5}$ approx. 5 nM). Fraction A from 60-day-old rats could be subfractionated by osmotic shock and sucrose gradient centrifugation to obtain three subfractions, two of which are enriched in axolemma and display Na⁺ chennel activity. The other subfraction is enriched in myelin and shows no Na⁺ channel actiivty. The plasma membrane preparations from young rats (up to 10 days) are devoid of myelin and are useful for studies of Na⁺ channel activity.     

Desangeloylshairidin,X-ray structure determination of a sesquiterpene lactone from Guillonea scabra

The structure and absolute configuration of desangeloylshairidin, a guaianolide isolated from Guillonea scabra, have been established by X-ray diffraction analysis. No conformational change was observed in its seven-membered ring between the crystal and deuterochloroform solution states.     

Synergistic effects of ethanol and temperature on yeast mitochondria

At temperatures lower than 37°C, the ethanol inhibition constant (Ki) for growth or fermentation inrho ⁺ cells of theSaccharomyces cerevisiae strain S288C was always higher (1.1M) than inrho ^– mutants (0.7M). At 37°C these differences disappeared, and both strains were equally inhibited by ethanol (Ki=0.7m). Mitochondrial activity can be inhibited by high ethanol concentration and temperature. In fact, the stronger inhibition by ethanol of therho ⁺ strain at 37°C was due to the fact that, under these conditions, this strain loses the advantage conferred by mitochondrial activity since the induction ofrho ^– cells in the population is very high. This does not result in an increase in the frequency ofrho ^– mutants because of the poor viability of these mutants in conditions of high temperature and ethanol. In consequence, S288C strain becomes as strongly inhibited by ethanol as therho ^– mutant strains. Differences in viability were not related to the fatty acids and ergosterol composition of the strain. In the presence of ethanol, bothrho ⁺ andrho ^– strains modified their lipids in the same way, but these changes did not improve their ethanol tolerance. They were not due to differences in adaptation to ethanol either, since after successive transfers in ethanol, growth () and fermentation () rates in therho ^– mutants were increasingly inhibited with time, whereas in the S288C strain inhibition of and by ethanol remained unaltered. Rather,rho ^– mutants are less viable thanrho ⁺ cells because of the inability of the former to respire. At 37°C the Ki increased to 0.9M ethanol either when mitochondrial from highly ethanol-tolerant wine yeasts were transferred torho ^– mutants of the strain S288C or when the mitochondria of strain S288C were preadapted by growing the strain in glycerol instead of glucose before it was cultivated in ethanol.     

Xp-duplications with and without sex reversal

Duplications in Xp including the DSS (dosage sensitive sex reversal) region cause male to female sex reversal. We investigated two patients from families with Xp duplications. The first case was one of two sisters with karyotype 46,XY, der(22), t(X;22)(p11.3;p11)mat and unambiguous female genitalia. The living sister was developmentally retarded, and showed multiple dysmorphic features and an acrocallosal syndrome. The second case was a boy with a maternally inherited direct duplication of Xp21.3-pter with the breakpoint close to the DSS locus. He had multiple abnormalities and micropenis, but otherwise unambiguous male genitalia. We performed quantitative Southern blot analysis with probes from Xp22.13 to p21.2 to define the duplicated region. Clinical, cytogenetic, and molecular data from both patients were compared with those of previously reported related cases. A comparison of the extragenital symptoms revealed no differences between patients with or without sex reversal. In both cases, the symptoms were non-specific. Among 22 patients with a duplication in Xp, nine had unambiguous female genitalia and a well-documented duplication of the DSS region. Two patients with duplication of DSS showed ambiguous external genitalia. From these data, we conclude that induction of testicular tissue may start in these patients, but that the type of genitalia depends on the degree of subsequent degeneration by a gene in DSS.