The interaction of calmodulin with amphiphilic peptides

Calmodulin has recently been shown to form exceptionally tight, calcium-dependent complexes with several natural peptides (Kdiss greater than 10(-7) M). These peptides were demonstrated to be capable of forming basic, amphiphilic alpha-helices. To further illustrate the importance of this structural feature for calmodulin binding, several other amphiphilic alpha-helical peptides were tested for their ability to bind calmodulin. To monitor complexes of high affinity (greater than 10(8) M-1), a new competition assay was devised with Sepharose 4B-conjugated melittin. Stoichiometries were assessed by electrophoresis and equilibrium size exclusion chromatography. Three peptides, which were designed to form idealized amphiphilic alpha-helices were tested. The basic peptides, N alpha-9-fluorenylmethoxycarboxyl-(FMOC)-(Leu-Lys-Lys-Leu-Leu-Lys-L eu)1 and FMOC-(Leu-Lys-Lys-Leu-Leu-Lys-Leu)2 bind calmodulin in a 1:1 complex with dissociation constants of 150 and 3 nM, respectively. The acidic peptide, FMOC-(Leu-Glu-Glu-Leu-Leu-Glu-Leu)2 failed to bind calmodulin, even at micromolar concentrations. Complex formation between calmodulin and the 14-residue basic peptide leads to an increase in the helicity of the complex which is attributed to an increase of about 50% in the helicity of the peptide. Calmodulin also interacts with the neutral alpha-helical peptide toxin delta-hemolysin. Concomitant with binding, the fluorescence maximum of the unique Trp residue increases 2-fold and is blue-shifted. A dissociation constant could not be unambiguously estimated though, since delta-hemolysin has a strong tendency to self-aggregate. The above data support our hypothesis that a basic, amphiphilic alpha-helix is a structural feature which underlies the calmodulin-binding properties common to a variety of peptides.     

Comparison of chloramphenicol acetyltransferase variants in staphylococci. Purification, inhibitor studies and N-terminal sequences.

Four electrophoretic variants of chloramphenicol acetyltransferase (types A, B, C and D) found in chloramphenicol-resistant staphylococci were purified by affinity chromatography. Michaelis constants and the kinetics of inactivation with a variety of reagents for the four variants are virtually identical. Their similar amino acid compositions and near identical N-terminal sequences suggest a high degree of overall sequence homology. The thiol-specific reagents 5,5'-dithiobis-(2-nitrobenzoic acid), 2-nitro-5-thiocyanobenzoic acid and 2,2'-dithiopyridine are without significant effect on enzyme activity, whereas 1-fluoro-2,4-dinitrobenzene, N-ethylmaleimide, p-chloromercuribenzoic acid, iodoacetamide, and, particularly, bromoacetyl-CoA and diethyl pyrocarbonate are potent inhibitors. Iodoacetate is not an inhibitor. The results of chemical modification studies on the four enzyme variants and the identification of 3-carboxymethylhistidine in acid hydrolysates of one variant (type C) after inactivation with iodoacetamide suggest that a unique histidine residue may be involved in the mechanism of catalysis.     

Assessing life cycle environmental impacts of inoculating soybeans in Argentina with Bradyrhizobium japonicum

The International Journal of Life Cycle Assessment - To estimate life cycle impacts from introducing the yield-enhancing inoculant containing the nitrogen-fixing bacterium Bradyrhizobium japonicum...     

Identification and characterization of 10 polymorphic microsatellite loci in the German cockroach,Blattella germanica

Primer sequences and initial characterization are presented for 10 microsatellite loci isolated from the German cockroach, Blattella germanica. In a sample of 30 individuals from a single population sample, all loci were polymorphic with two to 12 alleles segregating per locus and levels of observed heterozygosity ranging from 0.27 to 0.92. One locus showed a deficit of heterozygotes. Experimental conditions are described for polymerase chain reaction multiplexing, which enables the genotyping of eight loci in three electrophoretic runs consisting of one set of three and two sets of two markers. Seven primer sets cross‐amplify in the related Blattella asahinai.     

Introduction of a mutation in the shutter region of antithrombin (Phe77 --> Leu) increases affinity for heparin and decreases thermal stability

The shutter region of serpins consists of a number of highly conserved residues that are critical for both stability and function. Several variants of antithrombin with substitutions in this region are unstable and predispose the carrier to thrombosis. Although most mutations in the shutter region investigated to date are deleterious with respect to serpin stability and function, the substitution of Phe51 by Leu in alpha(1)-antitrypsin results in enhanced stability. Here, we have investigated the effects of introducing an analogous mutation into antithrombin (Phe 77 to Leu). The mutation did not affect the kinetics of interaction with proteases. Strikingly, however, the thermostability of the protein was markedly decreased, with the serpin displaying a 13 degrees C decrease in melting temperature as compared to wild-type recombinant antithrombin. Further studies revealed that in contrast to wild-type antithrombin, the mutant adopted the latent (inactive) conformation upon mild heating. Previous studies on shutter region mutations that destabilize antithrombin revealed that such variants possess enhanced affinity for both heparin pentasaccharide and full-length heparin. The N135A/F77L mutant had unchanged affinity for heparin pentasaccharide, but the affinity for full-length heparin was increased. We suggest that the Phe77Leu mutation causes conformational changes around the top of the D-helix in antithrombin, in particular, to the arginine 132 and 133 residues that may mediate additional antithrombin/heparin interactions. This paper also demonstrates that there are major differences between the shutter regions of antithrombin and alpha(1)-antitrypsin since a stabilizing mutation in antitrypsin has the converse effect in antithrombin.     

Reshaping of sandstone surfaces by cryptoendolithic cyanobacteria: bioalkalization causes chemical weathering in arid landscapes

We report a novel weathering mechanism in South African sandstone formations, where cryptoendolithic cyanobacteria induce weathering by substrate alkalization during photosynthesis. As a result, the upper rock part is loosened and then eroded away by physical forces such as wind, water, trampling. This special type of ‘exfoliation’ is widely distributed and affects the geomorphology of whole sandstone mountain ranges and outcrops across several biomes. We show, that this weathering type is initiated by bioalkalization because of the photosynthesis of cryptoendolithic (i.e. those organisms living in small tight open spaces between the sand grains) cyanobacteria causing pH values high enough to enhance silica solution in the cryptoendolithic zone. As modern cyanobacteria are the initial photoautotrophic colonizers of bare rocks in arid and semiarid landscapes, it is possible that they may also have played a significant role in shaping sandstone landscapes in the geological past.     

Linear and threshold-dependent expression of secondary sexual traits in the same individual: insights from a horned beetle (Coleoptera: Scarabaeidae)

Elaborate horns or horn‐like structures in male scarab beetles commonly scale with body size either (a) in a linear fashion with horn size increasing relatively faster than body size or (b) in a threshold‐dependent, sigmoid fashion; that is, males smaller than a certain critical body size develop no or only rudimentary horns, whereas males larger than the threshold size express fully developed horns. The development of linear vs. sigmoid scaling relationships is thought to require fundamentally different regulatory mechanisms. Here we show that such disparate regulatory mechanisms may co‐occur in the same individual. Large males of the south‐east Asian Onthophagus (Proagoderus) watanabei (Ochi & Kon) (Scarabaeidae, Onthophagini) develop a pair of long, curved head horns as well as a single thoracic horn. We show that unlike paired head horns in a large number of Onthophagus species, in O. watanabei the relationship between head horns and body size is best explained by a linear model. Large males develop disproportionately longer horns than small males, but the difference in relative horn sizes across the range of body sizes is small compared to other Onthophagus species. However, the scaling relationship between the thoracic horn and body size is best explained by a strongly sigmoid model. Only males above a certain body size threshold express a thoracic horn and males smaller than this threshold express no horn at all. We found a significant positive correlation between head and thoracic horn length residuals, contrary to what would be expected if a resource allocation tradeoff during larval development would influence the length of both horn types. Our results suggest that the scaling relationship between body size and horn length, and the developmental regulation underlying these scaling relationships, may be quite different for different horns, even though these horns may develop in the same individual. We discuss our results in the context of the developmental biology of secondary sexual traits in beetles. © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 83 , 473–480.     

Factors affecting early unplanned readmission of elderly patients to hospital.

A random sample of 133 elderly patients who had an unplanned readmission to a district general hospital within 28 days of discharge from hospital was studied and compared with a matched control sample of patients who were not readmitted. The total group was drawn from all specialties in the hospital, and by interviewing the patients, their carers, the ward sisters, and the patients'' general practitioners the factors causing early unplanned readmission for each patient were identified. Seven possible principal reasons were found: relapse of original condition, development of a new problem, carer problems, complications of the initial illness, need for terminal care, problems with medication, and problems with services. There were also contributory reasons, and it was usual for several of these to be present in each case. The unplanned readmission rate was 6%; the planned readmission rate was 3%. It was thought that unplanned readmission was avoidable for 78 (59%) patients. Patients in the study group and in the control group showed significant differences in certain characteristics--such as low income, previous hospital admission, already having nursing care, and admission by general practitioners--and this might help to identify patients who are likely to be readmitted in an emergency.