Side‐chain thioamides as fluorescence quenching probes

Thioamides, single atom oxygen‐to‐sulfur substitutions of canonical amide bonds, can be valuable probes for protein folding and protease studies. Here, we investigate the fluorescence quenching properties of thioamides incorporated into the side‐chains of amino acids. We synthesize and incorporate Fmoc‐protected, solid‐phase peptide synthesis building blocks for introducing N^ε‐thioacetyl‐lysine and γ‐thioasparagine. Using rigid model peptides, we demonstrate the distance‐dependent fluorescence quenching of these thioamides. Furthermore, we describe attempts to incorporate of N^ε‐thioacetyl‐lysine into proteins expressed in Escherichia coli using amber codon suppression.     

Midkine Is Expressed and Differentially Processed during Chronic Obstructive Pulmonary Disease Exacerbations and Ventilator-Associated Pneumonia Associated with Staphylococcus aureus Infection

Staphylococcus aureus is sometimes isolated from the airways during acute exacerbations of chronic obstructive pulmonary disease (COPD) but more commonly recognized as a cause of ventilator-associated pneumonia (VAP). Antimicrobial proteins, among them midkine (MK), are an important part of innate immunity in the airways. In this study, the levels and possible processing of MK in relation to S. aureus infection of the airways were investigated, comparing COPD and VAP, thus comparing a state of disease with preceding chronic inflammation and remodeling (COPD) with acute inflammation (that is, VAP). MK was detected in the small airways and alveoli of COPD lung tissue but less so in normal lung tissue. MK at below micromolar concentrations killed S. aureus in vitro. Proteolytic processing of MK by the staphylococcal metalloprotease aureolysin (AL), but not cysteine protease staphopain A (SA), resulted in impaired bactericidal activity. Degradation was seen foremost in the COOH-terminal portion of the molecule that harbors high bactericidal activity. In addition, MK was detected in sputum from patients suffering from VAP caused by S. aureus but less so in sputum from COPD exacerbations associated with the same bacterium. Recombinant MK was degraded more rapidly in sputum from the COPD patients than from the VAP patients and a greater proteolytic activity in COPD sputum was confirmed by zymography. Taken together, proteases of both bacteria and the host contribute to degradation of the antibacterial protein MK, resulting in an impaired defense of the airways, in particular, in COPD where the state of chronic inflammation could be of importance.     

Deletions of the N-terminal regions of the human melanocortin receptors

The non-homologous N-terminal regions of four human melanocortin (MC) receptors were truncated in order to investigate their putative participation in ligand binding. Eleven constructs were made, where different numbers of residues from the N terminus were deleted. These constructs were used for transient expression experiments in COS cells and analysed by ligand binding. The results show that 27, 25, 28, and 20 amino acids could be deleted from the N terminus of the human MC1, MC3, MC4 and MC5 receptors, respectively, including all potential N-terminal glycosylation sites in the MC1 and the MC4 receptors, without affecting ligand binding or expression levels. The results indicate that the N-terminal regions of the human MC1, MC3, MC4 and MC5 receptors, do not play an important role for the ligand binding properties of these receptors.     

Genetic studies on a tumor growth-inhibitory factor in serum of normal mice and its relationship to NK activity

It has been shown that normal mouse serum contains a tumor growth-inhibitory factor (GIF). and that strain-dependent levels of GIF correlate with mouse NK activity. To further analyze the genetic control of GIF we have studied the growth-inhibitory activity of normal mouse serum from 8 different mouse strains and their F1 hybrids. A sensitive method using a chromogenic substrate for an endogenous lysosomal enzyme was used to measure the inhibitory activity of normal mouse serum on the mouse B16 melanoma. The highest level of GIF was found in old mice, lower activity in serum of young animals and no activity in suckling mice. To compare the genetic control of GIF and NK, spleen NK activity against B16 as well as YAC-1 targets was measured in parallel in the same animals. Confirming previous results we found the H-2k strains CBA and C3H to have high levels of GIF as well as NK activity, while the strain A/Sn and the A congenic strain A.SW had low levels of both activities. Experiments with H-2d and H-2b strains, however, showed that GIF and NK had a different genetic control; thus the DBA/2 and Balb/c strains had considerably higher GIF activity than the C57B1 and Leaden strains, while the reverse was true for NK activity. In F1 hybrid crosses between strains with high and low activity, high activity was inherited as a dominant trait for both GIF and NK. A backcross analysis in (A X CBA) X A backcross mice, segregating for NK and GIF showed that the two activities did not cosegregate. These studies therefore demonstrate that GIF and NK activity are under different genetic control, and do not support any direct or simple relationship between GIF and NK cells.     

Habitat Heterogeneity,Coexistence and Habitat Utilization in Five Backswimmer Species (Notonecta spp.; Hemiptera,Notonectidae)

In this study, we present data on life history, distribution and coexistence among five backswimmer species, Notonecta glauca, N. lutea, N. maculata, N. obliqua and N. reuteri, from 72 waters in a study area on the Swedish west coast. Two temporal life history patterns were found; species reproducing in the autumn (N. reuteri and N. lutea) and those reproducing in the spring (N. glauca N. obliqua and N. maculata). Female backswimmers were generally larger than conspecific males, except for N. lutea, in which the sexes were similar in size. The sex ratio of N. glauca, N. lutea and N. reuteri did not differ from 1/1. The most abundant species was N. glauca (49% of the waters inhabited by backswimmers) and then N. obliqua (19%), N. maculata (14%), N. lutea (10%) and N. reuteri (8%). In all, 972 individuals were recorded. The species were sequenced, with reference to their habitat use (from species occurring in temporal habitats to more stable and complex ones), as follows; N. maculata – N. glauca – N. obliqua – N. reuteri – N. lutea. N. glauca had the widest habitat distribution pattern. Two species combinations in pools were most commonly found and the five species were never found together. Possible competition among adults, according to their habitat distribution, may occur for: N. glauca with all species; N. obliqua mainly with N. glauca; N. lutea mainly with N. reuteri; N. reuteri mainly with N. lutea and N. glauca; N. maculata mainly with N. glauca. If body size is an important factor in the competitive ability between individuals, not merely species but also sex and temporal aspects have to be considered. The most intense competition should then exist between N. lutea and N. reuteri females.     

The two types of alpha cells during the development of the guinea-pig pancreas

Summary The prenatal and postnatal development of the two types of pancreatic A cells was examined in guinea-pigs. The A₁ and A₂ cells appeared at about the same time and were first seen in 26 days old fetuses, while B cells could not be recognized earlier than on the 39th day of intrauterine life. In all age groups examined with quantitative methods (65 days old fetuses to 9 months old guinea-pigs) the proportion of parenchyma in the pancreatic gland appeared higher in the cauda than in the caput. In the 3 months and 9 months old animals the proportions of the different types of islet cells were also higher in cauda than in caput. In each case the total weights of the A₁, A₂ and B cell fractions showed a similar growth rate relative to that of the body weights. The results are discussed in the light of previous observations in man and rat.Supported by the United States Public Health Service (AM-05759-05), the Swedish Medical Research Council (12X-562-02; 12X-109-03) and the Medical Faculty of Uppsala.     

Antiferromagnetic exchange interaction in the two-iron-two-sulphur ferredoxin from the blue-green alga Spirulina maxima studied with a highly sensitive magnetic balance

1. A highly sensitive magnetic balance of the Faraday type is described. 2. The magnetic susceptibility of the oxidized and reduced forms of the two-iron-two-sulphur ferredoxin from the blue-green alga Spirulina maxima has been measured over a wide temperature range. 3. The results can be interpreted within a simple model involving antiferromagnetically coupled iron atoms at the active site. The coupling, expressed as --J, is estimated to be 182 +/- 20/cm and 98 +5/-10 /cm for the oxidized and reduced forms, respectively.