Conversion of type II procollagen to collagen in Vitro: Removal of the carboxy-terminal extension is inhibited by several naturally occurring amino acids,polyamines, and structurally related compounds

Chick embryo sterna, which actively synthesize type II procollagen, were pulse-labeled with radioactive proline; protein synthesis was then inhibited by unlabeled proline and cycloheximide. After the inhibition of protein synthesis, several amino acids, polyamines, or structurally related compounds were added to the incubation medium. The conversion of procollagen, first to two intermediates, pC-collagen and pN-collagen, and then to collagen, was monitored by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The addition of 50 mm β-alanine, arginine, asparagine, glutamine, hydroxylysine, lysine, or ornithine, as well as agmatine, ?-aminocaproic acid, S-2-aminoethylcysteine, cadaverine, canavanine, putrescine, or spermine clearly inhibited the removal of the carboxy-terminal extension and pC-collagen accumulated; the removal of the amino-terminal extension was not affected. The inhibition of the conversion was reversible and unaffected by fetal calf serum. The results suggest that the conversion of type II procollagen to collagen requires at least two separate proteinases for the removal of amino-terminal and carboxy-terminal extensions. The results further suggest that naturally occurring molecules may be used to modulate the rate of conversion of procollagen to collagen, and development of analogs of these compounds may provide the means to interfere with excessive deposition of collagen in diseases with tissue fibrosis.     

Production of cyclomaltodextrin glucanotransferase of Bacillus circulans var. alkalophilus ATCC21783 in B. subtilis

Summary The cyclomaltodextrin glucanotransferase (CGTase, E.C. 2.4.1.19) gene from an alkalophilic Bacillus circulans var. alkalophilus ATCC21783 was cloned into Escherichia coli and B. subtilis. When cloned from E. coli to B. subtilis, the entire insert containing the CGTase gene was, depending on the plasmid construction, either unstable or the recombinant B. subtilis did not secrete the enzyme in significant amounts. To achieve efficient enzyme production in B. subtilis, the gene was placed under the control of the B. amyloliquefaciens -amylase promoter. In one of the constructions, both the promoter and the signal sequence of the gene were replaced with those of B. amyloliquefaciens, whereas in another construction only the promoter area was exchanged. The recombinant B. subtilis clones transformed with these plasmid constructions secreted CGTase into the culture medium 14 times as much as did the parental strain in shake flask cultures. In fermentor cultures in an industrially feasible medium the enzyme production was substantially higher, yielding 1.2 g/l of CGTase, which is about 33 times the amount of the enzyme produced by the parental strain in corresponding fermentations. Both of the plasmid constructions were stable when grown over 50 generations without antibiotic selection.     

Incident Hip Fractures among Community Dwelling Persons with Alzheimer’s Disease in a Finnish Nationwide Register-Based Cohort

Background

Previous cohort studies have shown that persons with Alzheimer’s disease (AD) have a higher risk of hip fractures but recent data from large representative cohorts is scarce.

Methods

We investigated the association between AD and prevalent and incident hip fractures in an exposure-matched cohort study conducted in Finland 2002–2009 (the Medication and Alzheimer’s disease in 2005 study; MEDALZ-2005). The study population included all community-dwelling persons with verified AD diagnosis in Finland on December 31, 2005 and one matched comparison person per AD case (N = 56,186, mean age 79.9 (SD 6.8) years, range 42–101 years). The diagnosis of AD was extracted from a special reimbursement register. Data on hip fractures during 2002–2009 was extracted from the Finnish National hospital discharge register. Analyses of incident hip fractures (n = 2,861) were restricted to years 2006–2009.

Results

Persons with AD were twice as likely to have previous hip fracture in 2005 (odds ratio, 95% confidence interval 2.00, 1.82–2.20) than matched aged population without AD. They were also more likely to experience incident hip fracture during the four-year follow-up (hazard ratio, 95% confidence interval 2.57, 2.32–2.84, adjusted for health status, psychotropic drug and bisphosphonate use). The AD-associated risk increase decreased linearly across age groups. Although people with AD had higher risk of hip fractures regardless of sex, the risk increase was larger in men than women.

Conclusion

Findings from our nationwide study are in line with previous studies showing that persons with AD, regardless of sex or age, have higher risk of hip fracture in comparison to general population. Although there was some suggestion of effect modification by age or sex, AD was consistently associated with doubling of the risk of incident hip fracture.     

Tn10 generated deletions of the ompB locus of Escherichia coli K12

Abstract We have isolated a set of Tn 10 -generated deletions starting from the distal end of the ompR envZ operon of Escherichia coli K12. Most of the deletions removed both ompR and envZ genes or ended in ompR . These deletions exhibited an OmpC⁻ OmpF⁻ phenotype. One deletion removed only part of envZ and the strain was phenotypically OmpC⁻ OmpF^+/−. This deletion of the distal part of envZ did not affect osmoregulation of ompC . However, ompF osmoregulation appeared reversed. High osmolarity in the growth medium resulted in production of OmpF close to the wild-type level.     

Effects of training on the exercise-induced changes in serum amino acids and hormones

The purpose of this study was to examine power-type athletes to determine changes in amino acid and hormone concentrations in circulating blood following 2 different high-intensity exercise sessions before and after the 5-week training period. Eleven competitive male sprinters and jumpers performed 2 different running exercise sessions: a short run session (SRS) of 3 x 4 x 60 m (intensity of 91-95%) with recoveries of 120 and 360 seconds, and a long run session (LRS) with 20-second intervals (intensity of 56-100%) with recoveries of 100 seconds to exhaustion. The concentrations of serum amino acids, hormones, and lactate were determined from the blood samples drawn after an overnight fast and 10 minutes before and after both SRS and LRS. The average blood lactate concentrations were 12.7 +/- 1.6 mmol;pdL(-1) and 16.6 +/- 1.4 mmol;pdL(-1) (p < 0.01) following SRS and LRS, respectively. The average total running time was longer (p < 0.001) following LRS (164 +/- 20 seconds) than following SRS (91 +/- 8 seconds). The fasting levels of all amino acids decreased (p = 0.024; 19.4%) after the 5-week period, whereas an increase (p = 0.007; 24.5%) was observed in the fasting concentration of testosterone (TE). The exercise sessions induced no changes in the total sum of all amino acids, but significant increases or decreases were observed in single amino acids. When the range of the relative concentration changes before and after the training period was compared, significant decreases were found in valine (p = 0.048), asparagine (p = 0.029), and taurine (p = 0.030) following SRS. There were significant increases in the absolute hormonal concentration changes following LRS with TE (p = 0.002; 30.4%), cortisol (COR; p = 0.006; 12.0%), and in the TE/COR ratio (p = 0.047; 21.0%) but not in the concentration of growth hormone (GH). The results of the study indicate that the speed and strength training period strongly decreases the fasting concentrations of amino acids in the power-trained athletes in a good anabolic state with the daily protein intake of 1.26 g;pdkg(-1) body weight. At the same time the intensive lactic exercise session induces strong decreases, especially in valine, asparagine, and taurine.     

Leucine supplementation does not enhance acute strength or running performance but affects serum amino acid concentration

This study described the effect of leucine supplementation on serum amino acid concentration during two different exercise sessions in competitive male power athletes. The subjects performed a strength exercise session (SES; n = 16; 26 +/- 4 years) or a maximal anaerobic running exercise session (MARE; n = 12; 27 +/- 5 years) until exhaustion twice at a 7-day interval. The randomized subjects consumed drinks containing leucine (100 mg x kg/body weight before and during SES or 200 mg x kg/body weight before MARE) or placebo. Blood specimens taken 10 min before (B) and after (A) the sessions were analyzed for serum amino acids. In SES the concentration of leucine was distinctly higher in the leucine supplemented group than in the placebo group in both B (p < 0.001) and A (p < 0.001) samples. The leucine concentration decreased in placebo but not in the leucine supplemented group following the exercise session. Isoleucine (p = 0.017) and valine (p = 0.006) concentration decreased more in the leucine supplemented group than in placebo in A samples. In MARE the concentration of leucine was higher in the leucine supplemented group than in placebo in both B (p < 0.001) and A (p < 0.001) samples and increased (p < 0.001) in the supplemented group following the session. Isoleucine (p = 0.020) and valine (p = 0.006) concentration decreased in the supplemented group in A samples. There were no differences in a counter movement jump after SES or in the running performance in MARE between the leucine supplemented group and placebo. These findings indicate that consuming leucine before or before and during exercise sessions results in changes in blood amino acid concentration. However, the supplementation does not affect an acute physical performance.     

Primary Amine Oxidase of Escherichia coli Is a Metabolic Enzyme that Can Use a Human Leukocyte Molecule as a Substrate

Escherichia coli amine oxidase (ECAO), encoded by the tynA gene, catalyzes the oxidative deamination of aromatic amines into aldehydes through a well-established mechanism, but its exact biological role is unknown. We investigated the role of ECAO by screening environmental and human isolates for tynA and characterizing a tynA-deletion strain using microarray analysis and biochemical studies. The presence of tynA did not correlate with pathogenicity. In tynA+ Escherichia coli strains, ECAO enabled bacterial growth in phenylethylamine, and the resultant H₂O₂ was released into the growth medium. Some aminoglycoside antibiotics inhibited the enzymatic activity of ECAO, which could affect the growth of tynA+ bacteria. Our results suggest that tynA is a reserve gene used under stringent environmental conditions in which ECAO may, due to its production of H₂O₂, provide a growth advantage over other bacteria that are unable to manage high levels of this oxidant. In addition, ECAO, which resembles the human homolog hAOC3, is able to process an unknown substrate on human leukocytes.     

Genetic and environmental influence on maize kernel proteome

Comparative targeted compositional analysis is currently an important element in the safety assessment of genetically modified plants. Profiling methods have been suggested as nontargeted tools to improve the detection of possible unintended effects. In this study, the capability of 2-dimensional electrophoresis to detect significant differences among seven conventional maize (Zea mays) cultivars grown in six different locations in Germany during two consecutive seasons was evaluated. Besides maize genotype, both geographic location and season had a significant effect on protein profiles. Differences as high as 55- and 53-fold in the quantity of specific proteins were recorded, the median observed difference being around 6- and 5-fold between the genotypes and growing locations, respectively. Understanding the variation in the quantity of individual proteins should help to put the variation of endogenous proteins and the novel proteins in the genetically modified plants in perspective. This together with the targeted analyses the profiling methods, including proteomics, could also help to get a deeper insight into the unintended alterations that might have occurred during the genetic modification process.     

Specific recognition of malondialdehyde and malondialdehyde acetaldehyde adducts on oxidized LDL and apoptotic cells by complement anaphylatoxin C3a

Oxidatively modified low-density lipoproteins (Ox-LDL) and complement anaphylatoxins C3a and C5a are colocalized in atherosclerotic lesions. Anaphylatoxin C3a also binds and breaks bacterial lipid membranes and phosphatidylcholine liposomes. The role of oxidized lipid adducts in C3a binding to Ox-LDL and apoptotic cells was investigated. Recombinant human C3a bound specifically to low-density lipoprotein and bovine serum albumin modified with malondialdehyde (MDA) and malondialdehyde acetaldehyde (MAA) in chemiluminescence immunoassays. No binding was observed to native proteins, LDL oxidized with copper ions (CuOx-LDL), or phosphocholine. C3a binding to MAA-LDL was inhibited by two monoclonal antibodies specific for MAA-LDL. On agarose gel electrophoresis, C3a comigrated with MDA-LDL and MAA-LDL, but not with native LDL or CuOx-LDL. C3a bound to apoptotic cells in flow cytometry. C3a opsonized MAA-LDL and was taken up by J774A.1 macrophages in immunofluorescence analysis. Complement-activated human serum samples (n=30) showed increased C3a binding to MAA-LDL (P<0.001) and MDA-LDL (P<0.001) compared to nonactivated samples. The amount of C3a bound to MAA-LDL was associated with total complement activity, C3a desArg concentration, and IgG antibody levels to MAA-LDL. Proteins containing MDA adducts or MAA adducts may bind C3a in vivo and contribute to inflammatory processes involving activation of the complement system in atherosclerosis.