Purification and reaction mechanism of the primary inhibitor of plasmin from human plasma

The primary inhibitor of plasmin in human plasma was purified by a four-step procedure involving fractional (NH(4))(2)SO(4) precipitation, ion-exchange chromatography on a column of DEAE-Sepharose CL-6B and affinity chromatography on both a plasminogen-CH-Sepharose 4B column and a column of 6-aminohexanoic acid covalently coupled through the carboxylate function to AH-Sepharose 4B. No impurities in the final preparation could be detected when tested by immunoelectrophoresis against a range of specific antisera or against rabbit anti-human serum. On polyacrylamide-gel electrophoresis the inhibitor preparation showed a single band. The dissociation constant for the inhibitor-plasminogen complex was determined to be approx. 3mum at pH7.8. The reactions of the inhibitor with human plasmin and with bovine trypsin were studied. Comparison of the results obtained confirms the hypothesis previously presented, namely that the reaction of the inhibitor with plasmin involves at least two steps, the initial rapid formation of an enzyme-inhibitor complex followed by a slow irreversible transition to another complex. The results also indicate that the reaction of the inhibitor with trypsin involves just a single, irreversible step, so that this reaction seems to be less complicated than that of the inhibitor with plasmin. The ways in which 6-aminohexanoic acid influences the reactions were studied. The same value for the dissociation constant (approx. 26mum) for 6-aminohexanoic acid is obtained for both its effect on the reaction of the inhibitor with trypsin and for competitive inhibition of trypsin. The inhibitory effect of 6-aminohexanoic acid thus seems to be due to its blocking of the active site of trypsin. In contrast with this, the inhibitory effects of l-lysine and 6-aminohexanoic acid on the inhibitor-plasmin reaction occur at concentrations much too low to affect the active site of plasmin. The possible dependence of the reaction of the inhibitor with plasmin on a second site(s) on plasmin is discussed.     

Biometeorology in austria: past,present and future

Biometeorology in Austria has been shaped by concepts, personalities, and technology. In early times, the branches of biometeorology that are usual today were already evident: agricultural and forest meteorology, phenology, medical biometeorology and balneology, aerial biometeorology, urban housing and stabling meteorology all started to emerge several centuries ago. From the 1920 up to 1936, Wilhelm Schmidt at the Agricultural University of Austria laid the foundations of modern biometeorology. He was followed by Franz Sauberer, who headed a Department of Biometeorology at the National Weather Service and devoted his active life totally to biometeorology. Several years after his untimely death, the Department was dissolved. Not until 1981 was biometeorology taken up again at the Agricultural University, where the tradition of Schmidt and Sauberer now lives on in several courses within the area of applied biometeorology: Micro-and Topoclimatology, Agricultural and Forest Meteorology and Atmospheric Radiation. Biometeorology, being an experimental science, has also been influenced by new technological developments. The early period was exclusively observational. During the late nineteenth and early twentieth centuries mechanical and simple electric instruments were used with strip-chart recorders. These time consuming methods have now been replaced by electronic devices, including data loggers and portable computers along with many new electronic sensors, which provide additional insight into biometeorological problems. Since computers also make it possible to solve some of the complicated equations of biometeorology, the future of this science seems to be bright, not only in Austria but throughout the world.     

Purification and amino acid sequence of sakacin A, a bacteriocin from Lactobacillus sake Lb706.

Sakacin A, a bacteriocin produced by Lactobacillus sake Lb706 and which inhibits the growth of Listeria monocytogenes, was purified to homogeneity by ammonium sulphate precipitation and ion-exchange, hydrophobic-interaction and reversed-phase chromatography. The complete amino acid sequence of sakacin A was determined by Edman degradation. The bacteriocin consisted of 41 amino acid residues and had a calculated M(r) of 4308.7, which is in good agreement with the value determined by mass spectrometry. The structural gene encoding sakacin A (sakA) was cloned and sequenced. The gene encoded a primary translation product of 59 amino acid residues which was cleaved between amino acids 18 and 19 to yield the active sakacin A. Sakacin A shared some sequence similarities with other bacteriocins.     

A diaphragm diffusion cell applied to ethanol diffusion in agarose gel: A reproducibility study

Summary The effective diffusion coefficient of ethanol in a 4 %(w/v) agarose gel at 25°C was measured using a diaphragm diffusion cell. The reproducibility was very good; a standard deviation of only 2% was obtained. The mean value (9.5×10⁻⁶ cm²/s) agreed well with available theory and previous investigations.     

Biofilm build-up of Pseudomonas putida in a chemostat at different dilution rates

Summary A test system was set up where the build-up of a biofilm on a defined surface could be studied in a carbon source limited chemostat.The attachment of P. putida ATCC 11172 to glass when growing on L-asparagine was studied at different dilution rates (specific growth rates) from 0.1 to 1.5 h^–1 The number of attached colony forming units (cfu) increased with dilution rate from 1×10⁶ cfu/cm² at 0.1 h^–1 to 4×10⁷ cfu/cm² at 1.0 h^–1 and then the attachment decreased to about 6×10⁶ cfu/cm² at higher dilution rates (1.1–1.5 h^–1). The number of attached cfu was measured after 24 h exposure. The value of the maximum specific growth rate in batch culture was 0.6 h^–1.The total amount of attached cell-mass followed roughly the same pattern as the viable count.The viable count of the cells suspended in the growth medium showed its lowest value at the same dilution rate as resulted in maximum adhesion.It was shown that the effect of growth rate on the biofilm build-up of P. putida is significant, and ought to be borne in mind when continuous culture systems are set up and results evaluated.     

Dynamics of the cytoskeleton of epidermal cells in situ and in culture

Summary The cytoskeleton of primary tissue-culture cells from the epidermis of Xenopus laevis tadpoles was investigated by phase-contrast, immunofluorescence, and electron microscopy. The connection between the arrangement of different types of filaments and the mechanical properties of the epidermis is discussed. The bilayered epidermis attains stability from thick bundles of tonofilaments interconnecting the basal desmosomes. Twisting of tonofilaments around each other can explain the occurrence of elastic filamentous curls forming a meshwork braced between rows of small desmosomes in the apical region of the epidermis. Actin is arranged as a diffuse meshwork and sometimes forms bundles intermingling with tonofilament bundles. Surface membranes and rows of small desmosomes are delineated by actin and contain -actinin. Actin raises the tension for rounding and spreading of cells. Microtubules stabilize already well-developed lamellae.     

Localization and characterization of N-ethylmaleimide sensitive inhibitor(s) of thiol cathepsin activity from cultured nil and polyoma virus-transformed nil hamster cells

Exposure of cultured Nil (a stable line of fibroblast cells from Syrian hamsters) or polyoma virus-transformed (PyNil) hamster fibroblasts to 0.5 mM N-ethylmaleimide for 5 minutes resulted in striking increases in thiol cathepsin activity in unfractionated cell-free lysates. The paradoxical increase in activity of the normally N-ethylmaleimide-sensitive cathepsins apparently occurred as the result of the protective compartmentalization of the cathepsins in the lysosomes (20,000 X g sedimented fraction) and the unprotected localization of an inhibitor(s) in the soluble cytoplasm (175,000 X g supernatant fraction). Under continuous exposure of the cells to N-ethylmaleimide, a rapid increase in cathepsin activity (seen in the first 5 minutes) was followed by a steady decrease in activity (half inactivation time, 90 minutes). The relative difference in rates of N-ethylmaleimide inactivation of thiol cathepsins and thiol cathepsin inhibitors provides a means for estimating lysosomal cathepsin activity in whole cell extracts without the need for more time-consuming fractionation procedure. In reciprocal inhibition tests, it was found that, regardless of the source of cathepsins, the Nil and PyNil cathepsin inhibitor(s) inactivated the cathepsins to approximately the same extent. The inhibitors were heat stable (90-100 degrees C for 15 minutes) at pH 4, but were totally inactivated when boiled at pH 8.5. On a calibrated Sephadex G-100 column, the relative molecular weight (Mr) of the inhibitor(s) was 13,000 daltons. On the same column, the Mr of the cathepsins was 24,000 daltons. Compared with the cathepsin activity from Nil cells, there was about five times less cathepsin activity recoverable from the PyNil cells.     

Quantum yield and energy dependence for photooxidation of chlorophyllide in greening wheat

Dark grown leaves of wheat were irradiated with red light of different intensities, at a temperature close to 0°C. The rate of photoreduction of the protochlorophyllide 650-form into chlorophyllide 684-form was measured. On continued irradiation the chlorophyllide 684-form was photodecomposed. By comparing the rates of the two processes the quantum yield for photooxidation of the chlorophyllide 684-form was calculated. The quantum yield was 2°10^-5 at an intensity of 2200 W m^-2, and increased with decreasing light intensity to 3.2°10^-5 at an intensity of 170 W m^-2.     

Weather patterns and asthma epidemics in New York City and New Orleans,U.S.A.

Twenty-five years of data on asthmatic attacks in New Orleans (covering approximately 170,000 asthma attacks) have been analyzed to identify asthma epidemic days, defined as days on which an unusually high number of asthmatic individuals had attacks. Similar data covering three years was obtained for New York City. A preliminary examination of detailed meteoroligical data revealed a consistent meteorological pattern preceding and associated with such asthma epidemic days which consisted of a cold front preceding an asthma epidemic by one to three days followed by a high pressure system. The significance of these meteorological findings and their relationship to other environmental agents such as natural or man-made atmospheric pollutants that are likely to be associated with asthma attacks will be discussed.Presented at the Eighth International Congress of Biometeorology, 9–14 September 1979, Shefayim, Israel.