Biomedical subjects
Publications and source records attributed to D Walz.
Bovine platelet factor 4 has been crystallized by "vapor dilution" in space group P2(1)2(1)2(1), a = 63.7 A, b = 66.7 A, c = 80.5 A, with four molecules, each 9505 Mr, in the asymmetric unit. The crystals diffract X-rays to better than 2.8 A resolution.
In preparation for studies of the time course of production in vivo of the constituent chains of rabbit fibrin, we characterized the structural features of the fibrin molecule. Fibrin was isolated from plasma and reduced and alkylated. The alpha, beta, and gamma chains were separated by CM-cellulose chromatography and their molecular weights and amino acid compositions were determined. The gamma chain was sequenced 36 steps with 32 positive identifications and the beta chain, 12 steps with 12 identifications. No major differences between the sequences of these chains and those of man, chicken, and dog were noted.
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Injection of aqueous solutions of topically irritant substances intracutaneously into the skin of juvenile white mice creates a persisting oedema which can be assessed by the number of wrinkles formed upon reefing the skin at the site of injection. An algorithm is presented which transforms the number of wrinkles recorded just before as well as 2.5 h and 6 h after injection into the intensity of the oedematous reaction. The results obtained with a collective of about 10 mice per solution to be tested are reliable and reproducible; they conform to dose-response relations and hence can serve for quantifying local irritancy. The method is used to investigate the effect of pH, and a general model is developed describing the local irritancy of unphysiological pH-values.
By limited proteolysis with mouse submaxillaris protease, human prostatic acid phosphatase (EC 3.1.3.2) was cleaved into three fragments, Sp1, Sp2, and Sp3, which individually had no enzymatic activity. One of the fragments, Sp3, regained enzymatic activity after interaction with rabbit antibody to prostatic acid phosphatase. The Sp3 fragment was purified and characterized as to its molecular weight, amino acid composition, and carbohydrate content. The Sp3 fragment behaved like the parent molecule in L(+)-tartrate affinity and in trapping of a phosphoryl intermediate. The same Sp3 fragment also bears the most prominent antigenic determinants. This evidence suggest that Sp3 is the enzymatically active domain of prostatic acid phosphatase.
Isolated mitochondria suspended in an aerobic medium with 3-hydroxybutyrate or succinate serving as electron donor attain a stationary state with vanishing net flow of H+ ions (state 4). Adding valinomycin to such a suspension in the presence of various concentrations of K+ ions and a weak acid system such as acetate or phosphate creates new stationary states for the mitochondria which are characterized by a constant influx of K+ ions, while the net flow of H+ ions again vanishes due to the recycling of these ions by the weak acid system. Sufficiently low concentrations of K+ ions (less than 4 mM) cause these stationary states to last long enough for a separation of the mitochondria by centrifugation. The difference in electrochemical potential for H+ ions can then be determined by means of the partitioning of radioactively labelled markers. Suitable procedures to correct for binding of the markers are described. It is found that, for a constant affinity of the electron in the suspending medium, electron flow and the flow of K+ ions, which indicates the flow of pumped H+ ions, are linearly dependent on the electrochemical potential difference of H+ ions. The phenomenological coefficients obtained from these correlations are discussed with respect to the contributions of additive constants in the linear relations. It is found that, under the present experimental condition, such constants most likely vanish thus yielding symmetric flow-force relations. It is concluded that the redox-driven H+ pumps are not tightly coupled due to molecular slipping in the pumps and that the molecular stoichiometry is 2 H+ ions/electron for coupling site I and 4 H+ ions/electron for coupling sites II and III together.
The paper describes a method for judging the irritative potentialities of substances; proposes a scoring system with which the 'ordinary' risks of many of the abundant marketed products for washing, cleaning etc. may be predicted without biological testing; discusses pharmacodynamic and pharmacokinetic aspects of topical irritancy and points to the problems of extrapolating from animal experiments to man.
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The effect of antimycin A and funiculosin, two inhibitors which block electron transfer in the b-c1 complex, on electron flow and electrochemical potential difference of H+ ions in mitochondria at static head (state 4) is investigated. In addition, the respiratory control ratio is determined as the ratio between uncoupler stimulated and static-head electron flow. Malonate, a competitive inhibitor or succinic dehydrogenase, is used for comparison. All three inhibitors cause an extensive depression of static-head electron flow but only a limited decrease in the electrochemical potential difference of H+ ions. With the antimycin-type of inhibitors, the respiratory control ratio slightly increases up to about 50% inhibition of electron flow and then steeply declines. With malonate, a strong decrease of the respiratory control ratio is observed in a concentration range where the electron flow is inhibited less than 10%. It is shown than the data do not comply with the generally accepted hypothesis of a leak conductance being regulated by the electrochemical potential difference of H+ ions. They can be interpreted in terms of not tightly coupled redox-driven H+-pumps. A non-vanishing electron flow at static head then arises predominantly from molecular slipping in the pumps, and the (constant) leak conductance yields only a minor contribution.
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The abdominal skin of juvenile white mice was used. Topical application was accomplished by intracutaneous injection. The solutions tested were thoroughly standardized. By using a special buffer system, a mixture of histidine glutamate and lysine glutamate, each pH value could be tested with the same buffer capacity. The pH values varied from pH 3 to pH 11, the solutions being always iso-osmolalic with plasma. Two criteria were used to judge the irritancy: (a) the edematous reaction for the first 6 h and (b) the macroscopic aspect after 24 h. A very special procedure enabled us to obtain objective numerical values for criterion (a). The results disclose that irritation becomes manifest at pH 4 and pH 10, becomes clear-cut with pH 3 and even more so with pH 11, and depends strongly on the buffer capacity employed. With two substances-amidosulfuric acid and (tri) sodium phosphate--it was exemplified how the procedure could be used to routinely screen for topical irritancy in an informative, inexpensive and decent manner.
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Vesicles obtained by sonication of chlorophyll a-lecithin mixtures dispersed in anaqueous medium closely resemble the well-characterized vesicles similarly prepared from pure lipids. They are bounded by one spherical lipid bilayer which contains the chlorophyll a. Appropriate conditions for sonication prevent substantial degradation of the membrane constituents. Up to one chlorophyll a molecule per 55 lecithins can be incorporated into membranes. The average Stokes' radius of the vesicles determined by analytical sieve chromatography is 102 +/- 5 A and independent of the chloropyll a content. The membrane is visible in the electron-microscope when the vesicles are treated with osmium tetroxide prior to negative staining. The osmium fixation is, however, not strong enough to allow for a preparation of the vesicles for thin sectioning (dehydration, embedding in epoxide).
Valinomycin added to a suspension of chlorophyll a containing lecithin vesicles induces slight changes in the spectrum of chlorophyll a. These changes are measured as a difference spectrum between samples with and without valinomycin but of otherwise identical composition. The analysis of the experiments reveals that the effect is neither associated with the ionophoric properties of valinomycin nor due to a direct interaction of this agent with chlorophyll a. The molar ratio of valinomycin dissolved in the membrane to lecithin is found to be the relevant parameter, thus indicating an interaction between these two components. As a consequence, the aggregational state of the lecithin molecules is altered. Chlorophyll a incorporated into the membrane acts as a sensor, i.e. it reflects the alteration by a change in its spectroscopic parameters.
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