Purification of smooth-muscle myosin free of calmodulin and myosin light-chain kinase. Susceptibility to oxidation.

Smooth-muscle myosin purified as described by Persechini & Hartshorne [(1983) Biochemistry 22, 470-476] contains trace amounts of calmodulin and myosin light-chain kinase, which can be removed by Ca2+-dependent hydrophobic-interaction chromatography followed by calmodulin-Sepharose affinity chromatography. The resultant column-purified myosin exhibits properties similar to those of the non-purified myosin, e.g. actin activation of the Mg2+-ATPase requires Ca2+/calmodulin-dependent phosphorylation of the two 20 kDa light chains. However, unlike the non-purified myosin, the column-purified myosin undergoes a time-dependent transition to a form which no longer requires phosphorylation for actin activation of the myosin Mg2+-ATPase. This transition is identified as a time-dependent change in conformation of the column-purified myosin from a 10 S to 6 S form and is caused by slow oxidation of the column-purified myosin, since it could be prevented by storage under N2 and reversed by 5 mM-dithiothreitol.     

Fluorescence technique for comparative studies of substrate-binding subsites in serine proteinases. Application to subtilisins.

A fluorescence technique for comparative studies of substrate-binding subsites in serine proteinases is described. It consists of: selective labelling of the corresponding subsites with a fluorescent group by using N alpha-dansyl(5-dimethylaminonaphthalene-1-sulphonyl)ated peptide chloromethanes containing different numbers of amino acid residues, and probing the immediate environment of the subsites by quenching experiments using ionic and neutral quenchers. Intramolecular distances between the subsites and particular chromophores can be also determined. The technique is of general applicability to all serine proteinases. The above mentioned approach was applied to two proteinases: subtilisin Novo and mesentericopeptidase. It was concluded that the substrate-binding site of mesentericopeptidase is considerably more polar than that of subtilisin Novo. Intramolecular distances between the labelled subsites and tryptophan residues in the two proteinases were determined.     

Nutrient conservation strategies in native Andean‐Patagonian forests

Abstract. Nutrient conservation in vegetation affects rates of litter decomposition and soil nutrient availability. Although resorption has been traditionally considered one of the most important plant strategies to conserve nutrients in temperate forests, long leaf life‐span and low nutrient requirements have been postulated as better indicators. We aimed at identifying nutrient conservation strategies within characteristic functional groups of NW Patagonian forests on Andisols. We analysed C‐, N‐, P‐, K‐ and lignin‐concentrations in mature and senescent leaves of ten native woody species within the functional groups: broad‐leaved deciduous species, broad‐leaved evergreens and conifers. We also examined mycorrhizal associations in all species. Nutrient concentration in mature leaves and N‐ resorption were higher in broad‐leaved deciduous species than in the other two functional groups. Conifers had low mature leaf nutrient concentrations, low N‐resorption and high lignin/N ratios in senescent leaves. P‐ and K‐resorptions did not differ among functional groups. Broad‐leaved evergreens exhibited a species‐dependent response. Nitrogen in mature leaves was positively correlated with both N resorption and soil N‐fertility. Despite the high P‐retention capacity of Andisols, N appeared to be the more limiting nutrient, with most species being proficient in resorbing N but not P. The presence of endomycorrhizae in all conifers and the broad‐leaved evergreen Maytenus boaria, ectomycorrhizae in all Nothofagus species (four deciduous, one evergreen), and cluster roots in the broad‐leaved evergreen Lomatia hirsuta, would be possibly explaining why P is less limiting than N in these forests.     

Clinico-epidemiological characteristics of Q fever in the Carpathian region

The study of the specific epidemiological and clinical features of Q fever revealed the existence of an active focus of infection among humans due to their contacts with agricultural animals in one of the districts of the region. The focus was manifested by group morbidity among the cattle-tending personnel of a dairy farm. The source of this infection was cattle. The infection was transferred mainly through the air. The disease took a moderately severe course. The study of the rickettsial contamination of humans, animals and ticks suggested the presence of the active epidemic process and made it possible to work out concrete antiepidemic measures.     

A specific decrease in collagen synthesis in acutely fasted, vitamin C-supplemented, guinea pigs

Weight loss often results from various experimental conditions including scurvy in guinea pigs, where we showed that decreased collagen synthesis was directly related to weight loss, rather than to defective proline hydroxylation (Chojkier, M., Spanheimer, R., and Peterkofsky, B. (1983) J. Clin. Invest. 72, 826-835). In the study described here, this effect was reproduced by acutely fasting normal guinea pigs receiving vitamin C, as determined by measuring collagen and non-collagen protein production after labeling tissues in vitro with [3H]proline. Collagen production (dpm/microgram of DNA) decreased soon after initiating fasting and by 96 h it had reached levels 8-12% of control values. Effects on non-collagen protein were much less severe, so that the percentage of collagen synthesis relative to total protein synthesis was 20-25% of control values after a 96-h fast. These effects were not due to changes in the specific radioactivity of free proline. Refeeding reversed the effects on non-collagen protein production within 24 h, but collagen production did not return to normal until 96 h. The effect of fasting on collagen production was independent of age, sex, ascorbate status, species of animal, and type of connective tissue and also was seen with in vivo labeling. Pulse-chase experiments and analysis of labeled and pre-existing proteins by gel electrophoresis showed no evidence of increased collagen degradation as a result of fasting. Procollagen mRNA was decreased in tissues of fasted animals as determined by cell-free translation and dot-blot hybridization with cDNA probes. In contrast, there was no decrease in translatable mRNAs for non-collagen proteins. These results suggest that loss of nutritional factors other than vitamin C lead to a rapid, specific decrease in collagen synthesis mainly through modulation of mRNA levels.     

Influence of chronic antigen exposure on the metabolism of PGH2 by microsomal preparations of airway and parenchymal tissues in the sheep

The effects of repeated antigen exposure on the synthesis of mediators by lung tissues are not well understood. To investigate the influence of antigen challenge on the synthesis of prostaglandins by central airway and peripheral lung tissues, fourteen sensitive sheep underwent biweekly exposure to aerosolized $\text{Ascaris suu}$ antigen (7) or saline (7). Following the fifth exposure, microsomal and high speed supernatant fractions were prepared from trachealis muscle and lung parenchyma. Synthesis of thromboxane (TX) A₂, prostaglandin (PG) D₂ and PGI₂ from the PG endoperoxide intermediate, PGH₂, was assayed over a range of substrate concentrations from 3–200 uM. Synthesis of PGI₂ by trachealis microsomes was approximately 5-fold greater than that of TXA₂. PGI₂ and TXA₂ production was identical in tracheal preparations from Ascaris- and saline-exposed animals. In parenchymal tissues, where TXA₂ production predominated over PGI₂ by 9-fold, preparations from Ascaris- exposed animals synthesized 50% more TXA₂ than controls at PGH₂ concentrations of 25 uM and above, whereas synthesis of PGI₂ and PGD₂ were similar in preparations from both groups of animals. The density of pulmonary mast cells was decreased by 21% in the Ascaris group, whereas polymorphonuclear leukocyte density was unchanged. These results demonstrate the differential synthesis of TXA₂ and PGI₂ in central airways and peripheral lung regions of the sheep. They further indicate that repeated exposure of the airways to antigen selectively enhances TXA₂ synthesis in the lung periphery of sensitized animals. The site of this increased enzymatic activity, whether in resident cells or newly-infiltrated cells, has not been determined.