C5a fragment of bovine complement. Purification, bioassays, amino-acid sequence and other structural studies

C5a and des-Arg-C5a have been purified from bovine serum in milligram amounts. The progress of the purification was followed by measuring the chemotactic activity of the complement fragments. The two polypeptides induce activation of neutrophil-oriented locomotion and secretion with very similar dose/response effects. After preparing a rabbit antiserum to bovine C5a/des-Arg-C5a, a competitive enzyme-linked immunosorbent assay (ELISA) was set up for the detection of C5a from 5 ng/mol to 1 microgram/ml. The complete primary structure of bovine C5a, which consists of 74 amino acids, has been determined by sequence analysis of the tryptic peptides, aligned by peptides derived from a chymotryptic digest, and by partially sequencing the intact molecule. Bovine C5a has a sequence homology of 78% and 70% with porcine and human C5a, respectively, reacts with an antiserum to porcine C5a and is recognized by cell surface receptors on human neutrophils. Finally, the secondary structure of bovine C5a was investigated by circular dicroic spectroscopy and predicted from the amino acid sequence. A comparison of the content and distribution of alpha-helical and/or hydropathic regions, suggests that the three-dimensional structure of C5a might be modeled from the known crystal structure of the homologous C3a molecule.     

Effects of scarcity and excess of larval food on life history traits of Aedes aegypti (Diptera: Culicidae)

Few studies have assessed the effects of food scarcity or excess on the life history traits of Aedes aegypti (L.) (Diptera: Culicidae) independently from larval density. We assessed immature survival, development time, and adult size in relation to food availability. We reared cohorts of 30 Ae. aegypti larvae from newly hatched to adult emergence with different food availability. Food conditions were kept constant by transferring larvae each day to a new food solution. Immature development was completed by some individuals in all treatments. The shortest development time, the largest adults, and the highest survival were observed at intermediate food levels. The most important effects of food scarcity were an extension in development time, a decrease in the size of adults, and a slight decrease in survival, while the most important effects of food excess were an important decrease in survival and a slight decrease in the size of adults. The variability in development time and adult size within sex and treatment increased at decreasing food availability. The results suggest that although the studied population has adapted to a wide range of food availabilities, both scarcity and excess of food have important negative impacts on fitness.     

Influence of supplemental ultraviolet-B on indoleacetic acid and calmodulin in the leaves of rice (Oryza sativa L.)

IR68 and Dular rice cultivars were grown under ambient, 13.0 (simulating 20% ozone depletion) and 19.1 (simulating 40% ozone depletion) kJ m^-2 day^-1 of biologically effective ultraviolet-B (UV-B_BE) for 4 weeks. Plant height and leaf area were significantly reduced by supplemental UV-B_BE radiation. Greater reduction in leaf area than of plant height was observed. A decrease in indole-3-acetic acid (IAA) content and increase in peroxidase and IAA oxidase activities of UV-B treated plants in both cultivars were observed compared with ambient control. Calmodulin content also decreased after plants were treated with high supplemental UV-B for two weeks and medium UV-B treatment for four weeks. The results indicated that peroxidase and IAA oxidase activities in rice leaves were stimulated by supplemental UV-B, resulting in the destruction of IAA which in turn may cause inhibition of rice leaf growth. Although the mechanism is unclear, calmodulin is most likely involved in leaf growth.     

Effects of fatigue on sarcoplasmic reticulum and myofibrillar properties of rat single muscle fibers.

Force decline during fatigue in skeletal muscle is attributed mainly to progressive alterations of the intracellular milieu. Metabolite changes and the decline in free myoplasmic calcium influence the activation and contractile processes. This study was aimed at evaluating whether fatigue also causes persistent modifications of key myofibrillar and sarcoplasmic reticulum (SR) proteins that contribute to tension reduction. The presence of such modifications was investigated in chemically skinned fibers, a procedure that replaces the fatigued cytoplasm from the muscle fiber with a normal medium. Myofibrillar Ca(2+) sensitivity was reduced in slow-twitch muscle (for example, the pCa value corresponding to 50% of maximum tension was 6.23 +/- 0.03 vs. 5.99 + 0.05, P < 0.01, in rested and fatigued fibers) and not modified in fast-twitch muscle. Phosphorylation of the regulatory myosin light chain isoform increased in fast-twitch muscle. The rate of SR Ca(2+) uptake was increased in slow-twitch muscle fibers (14.2 +/- 1.0 vs. 19.6 +/- 2. 5 nmol. min(-1). mg fiber protein(-1), P < 0.05) and not altered in fast-twitch fibers. No persistent modifications of SR Ca(2+) release properties were found. These results indicate that persistent modifications of myofibrillar and SR properties contribute to fatigue-induced muscle force decline only in slow fibers. These alterations may be either enhanced or counteracted, in vivo, by the metabolic changes that normally occur during fatigue development.     

Trapping sand flies (Diptera: Psychodidae) in the Emilia-Romagna region of northern Italy.

The efficiency and practicality of two trapping methods for adult Phlebotomine sand flies in two areas of the Emilia-Romagna region (Italy) were evaluated. Suction traps (CO2) and sticky traps (ST) were used to collect sand flies every two weeks, from June to September, 1999, from 16:00 to 07:00. Two CO2 traps were activated at the same time for each area (one with light and one without light), whereas 38 (four with light and 34 without lights) and 48 (four with light and 44 without) sticky traps were activated in Borghi and Longiano, respectively. An Index of Apparent Abundance (IAA) was calculated for each trap type and area. A total of 2,253 sand flies was trapped over the four-month period, with 1,765 collected from Borghi and 488 from Longiano. Phlebotomus perfiliewi was the most abundant species collected, comprising 99.6% and 84.6% of the total flies trapped in Borghi and Longiano, respectively. Other species were also collected within the two areas (Phlebotomus perniciosus and Phlebotomus mascittii) but were not considered for further analyses due to low catches. Significantly more specimens were caught using CO2 than sticky traps and the addition of a light source also improved the catches, however, a significantly greater number of female specimens were collected by a CO2 trap without a light source. Phlebotomus perfiliewi thus appears to show a photophobic reaction in the case of females when trapped using CO2/light attractants.     

Kinetic and enzymatic features of metabolic stimulation of alveolar and peritoneal macrophages challenged with bacteria

The rate of oxygen uptake and of ¹⁴C-1-glucose oxidation by peritoneal and alveolar macrophages has been simultaneously recorded before and after the exposure of the cells to B. mycoides. A stimulation of both processes was detected within seconds after the addition of bacteria. A comparison of ¹⁴C-1-glucose with ¹⁴C-6-glucose oxidation has indicated that the stimulation of the ¹⁴CO₂ production from ¹⁴C-1-glucose is substantially to be ascribed to an increased activity of the HMP pathway. On approaching anaerobiosis, the rate of the HMP pathway fell to zero, showing a direct link between cell respiration and production of NADP⁺ for the pathway. The assay of an enzyme, catalysing the reaction: NADPH + H⁺ + O₂ → NADP⁺ + H₂O₂, in 20 000 g pellets has shown that this oxidase has a higher activity in subcellular fractions derived from macrophages previously exposed to bacteria. The activation of this enzyme may be the most important event in the metabolic stimulation of macrophages challenged with bacteria. On the basis of experiments carried out with KCN, an inhibitor of both NADPH oxidase and catalase, it has been concluded that, under particular conditions, also the concerted action of GSH peroxidase and GSSG reductase might contribute to supporting the HMP pathway activity.     

Absence of coenzyme Q10 as an intrinsic component of aldehyde oxidase

Many investigators have purified an aldehyde oxidase from mammalian livers, and described reactions of this enzyme with diverse substrates. Coenzyme Q₁₀ was unambiguously identified and found present in some of these enzyme preparations. It was considered that coenzyme Q₁₀ might participate in the functionality of this enzyme, but the validity of the intrinsic association of coenzyme Q₁₀ was questioned. We have similarly purified aldehyde oxidase from rabbit livers. No coenzyme Q₁₀ could be detected under controlled conditions for detecting the presence of coenzyme Q₁₀. It is concluded that coenzyme Q₁₀ may be a contaminant of some aldehyde oxidase preparations, and that it is not intrinsic for the functionality of this enzyme.