Duplication of the CYP21A2 gene complicates mutation analysis of steroid 21-hydroxylase deficiency: characteristics of three unusual haplotypes

Steroid 21-hydroxylase deficiency, the primary cause of congenital adrenal hyperplasia, is caused by defects of the CYP21A2 gene. As a complement to hormonal measurements, mutation analysis of CYP21A2 is an important tool in the diagnosis of steroid 21-hydroxylase deficiency. Contemporary mutation-detection protocols based on the polymerase chain reaction often depend on the assumption that no more than one CYP21A2 gene is present on each chromosome 6. We describe three haplotypes with two CYP21A2 genes on the same chromosome, with defects typical of salt-losing steroid 21-hydroxylase deficiency in one of those genes, but not necessarily in the other. The frequency of these haplotypes in the general population is 6/365 (1.6%), so they are no less common than other haplotypes that indeed carry steroid 21-hydroxylase deficiency. Chromosomes that carry two CYP21A2 genes therefore represent a significant pitfall in the molecular diagnosis of steroid 21-hydroxylase deficiency. We recommend that, whenever CYP21A2 mutation analysis of an individual who is not a known carrier of steroid 21-hydroxylase deficiency is performed, the overall structure of the CYP21/ C4 region (the RCCX area) is determined by haplotyping to avoid erroneous assignment of carrier status.     

Targeting of peptides to restenotic vascular smooth muscle cells using phage display in vitro and in vivo

Restenosis after angioplasty occurs in 30-40% of the treated patients. To develop a strategy to deliver drugs to restenotic lesions, we selected phages that bind to proliferating vascular smooth muscle cells (VSMC), from a random constraint 15-mer peptide phage display library. Phages were selected for binding to cultured primary aortic VSMC (in vitro biopanning) and selected for binding to denudated carotid arteries in mice (in vivo biopanning). In vitro biopanning did not result in a consensus sequence, but recurring FLGW and LASR amino acid motifs were identified. In vivo biopanning resulted in two consensus peptides 5G6 (CNIWGVVLSWIGVFPEC) and 5E5 (CESLWGGLMWTIGLSDC). Surprisingly, these two sequences were recovered after both in vitro and in vivo biopanning, but predominantly in vivo. Moreover, a strong recurring motif, IGR, was identified in the in vivo clones. The consensus phages 5G6 and 5E5 bind selectively to VSMC compared to other cell types. Furthermore, they bind preferentially to proliferating VSMC compared to VSMC that were growth arrested, and are effectively internalized by their target cells. The specific binding capacities of 5G6 and 5E5 phages suggest that these peptide sequences can be used for targeting of restenotic lesions, in which proliferating VSMC are the dominant cell type.     

Inertial shear forces and the use of centrifuges in gravity research. What is the proper control?

Centrifuges are used for 1 x g controls in space flight microgravity experiments and in ground based research. Using centrifugation as a tool to generate an Earth like acceleration introduces unwanted inertial shear forces to the sample. Depending on the centrifuge and the geometry of the experiment hardware used these shear forces contribute significantly to the total force acting on the cells or tissues. The inertial shear force artifact should be dealt with for future experiment hardware development for Shuttle and the International Space Station (ISS) as well as for the interpretation of previous space-flight and on-ground research data.     

Efficient production of Arthromyces ramosus peroxidase by Aspergillus awamori

The heterologous production of Arthromyces ramosus peroxidase (ARP) was analysed in the filamentous fungus Aspergillus awamori under control of the inducible endoxylanase promoter. Secretion of active ARP was achieved up to 800 mg l(-1) in shake flask cultures. Western blot analysis showed that an rARP product of the correct molecular weight was produced. In contrast to several other studies about heterologous production of heme containing peroxidases, our results suggest that in A. awamori no heme limitation exists during overproduction of ARP.     

Sequence, distribution and quantification of the motilin precursor in the cat

Due to motilin's relation to the migrating motor complex (MMC), the physiology of motilin has been mostly studied in man and dog. The cat does not have an MMC pattern, and little is known about cat motilin. Therefore we identified the cat motilin precursor (GenBank accession no. AF127917) and developed a quantitative polymerase chain reaction (PCR) to explore its distribution in the gastrointestinal tract and in the central nervous system (CNS). The precursor is closely related to the dog precursor and consists of an open reading frame of 348bp encoding the signal peptide (25 amino acids), the motilin sequence (22 amino acids) and the motilin associated peptide (69 amino acids). One amino acid of the signal peptide was subject to gene polymorphism. Quantification of motilin messenger RNA (mRNA) was for the first time achieved. It is most abundant in the gastrointestinal tract, with the highest concentration in the duodenum, the lowest in the colon and is not detectable in the corpus. However an important expression was also observed in several regions of the CNS, except the striatum and cerebral cortex. The highest level was in the hypothalamus (although 23-fold lower than in the duodenum), the lowest level in the pons. Moderate levels were found in the thyroid. These data suggest that the physiological role of motilin may extend beyond its effect on gastrointestinal motility.     

Cofactor Tpr2 combines two TPR domains and a J domain to regulate the Hsp70/Hsp90 chaperone system

In the eukaryotic cytosol, Hsp70 and Hsp90 cooperate with various co-chaperone proteins in the folding of a growing set of substrates, including the glucocorticoid receptor (GR). Here, we analyse the function of the co-chaperone Tpr2, which contains two chaperone-binding TPR domains and a DnaJ homologous J domain. In vivo, an increase or decrease in Tpr2 expression reduces GR activation, suggesting that Tpr2 is required at a narrowly defined expression level. As shown in vitro, Tpr2 recognizes both Hsp70 and Hsp90 through its TPR domains, and its J domain stimulates ATP hydrolysis and polypeptide binding by Hsp70. Furthermore, unlike other co-chaperones, Tpr2 induces ATP-independent dissociation of Hsp90 but not of Hsp70 from chaperone-substrate complexes. Excess Tpr2 inhibits the Hsp90-dependent folding of GR in cell lysates. We propose a novel mechanism in which Tpr2 mediates the retrograde transfer of substrates from Hsp90 onto Hsp70. At normal levels substoichiometric to Hsp90 and Hsp70, this activity optimizes the function of the multichaperone machinery.     

Time related changes in calcium handling in the isolated ischemic and reperfused rat heart

The main aim of this study was to assess the kinetics of intracellular free calcium (Ca²⁺ _i) handling by isolated rat hearts rendered ischemic for 30 min followed by 30 min of reperfusion analyzing the upstroke and downslope of the Ca²⁺ _i transient. Changes in mechanical performance and degradation of membrane phospholipids – estimated by tissue arachidonic acid content – were correlated with Ca²⁺ _i levels of the heart. The fluorescence ratio technique was applied to estimate Ca²⁺ _i. The disappearance of mechanical activity of the heart preceded that of the Ca²⁺ _i transient in the first 2 min of ischemia. The slope of upstroke of the Ca²⁺ _i transient, reflecting Ca²⁺ release, decreased by 60%, while the duration of the downslope of the transient, reflecting Ca²⁺ sequestration, expressed a significant prolongation (105 ± 17 vs. 149 ± 39 msec) during the first 3 min of ischemia. At about 20 min of ischemia end-diastolic pressure expressed a 3.5-fold increase (contracture) when the fluorescence ratio showed a 2-fold elevation. Reperfusion was accompanied with a further precipitous increase in end-diastolic pressure, while resting Ca²⁺ _i remained at end-ischemic levels. Increases in the arachidonic acid (AA) content of the ischemic and postischemic hearts were proportional to Ca²⁺ _i levels. In summary, the present findings indicate that both calcium release and removal are hampered during the early phase of ischemia. Moreover, a critical level of Ca²⁺ _i and a critical duration of ischemia may exist to provoke contracture of the heart. Upon reperfusion the hearts show membrane phospholipid degradation and signs of stunning exemplified by elevated AA levels, partial recovery of Ca²⁺ _i handling and sustained depression of mechanical performance.     

Inhibition of photosystem II (PSII) electron transport as a convenient endpoint to assess stress of the herbicide linuron on freshwater plants

Effects of the herbicide linuron on photosynthesis of the freshwater macrophytes Elodea nuttallii (Planchon) St. John, Myriophyllum spicatum L., Potamogeton crispus L., Ranunculus circinatus Sibth., Ceratophyllum demersum L. and Chara globularis (Thuill.), and of the alga Scenedesmus acutus Meyen, were assessed by measuring the efficiency of photosystem II electron flow using chlorophyll fluorescence. In a series of single-species laboratory tests several plant species were exposed to linuron at concentrations ranging from 0 to 1000 μg l−1. It was found that the primary effect of linuron, inhibition of photosystem II electron flow, occurred with a half-lifetime of about 0.1 to 1.9 h after addition of linuron to the growth medium. The direct effect of the herbicide on photosynthesis appeared to be reversible. Complete recovery from the inhibition occurred with a half-lifetime of 0.5 to 1.8 h after transfer of linuron treated plants to linuron free medium. The EC50,24h of the inhibition of photosystem II electron transport by linuron was about 9–13 μg l−1 for most of the macrophytes tested. For S. acutus the EC50,72h for inhibition of photosystem II electron flow was about 17 μg l−1 for the free suspension, and 22 μg l−1 for cells encapsulated in alginate beads. In a long-term indoor microcosm experiment, the photosystem II electron flow of the macrophytes E. nuttallii, C. demersum and the alga Spirogyra sp. was determined during 4 weeks of chronic exposure to linuron. The EC50,4weeks for the long-term exposure was 8.3, 8.7 and 25.1 μg l−1 for E. nuttallii, C. demersum and Spirogyra, respectively. These results are very similar to those calculated for the acute effects. The relative biomass increase of E. nuttallii in the microcosms was determined during 3 weeks of chronic exposure and was related to the efficiency of photosystem II electron transport as assessed in the different treatments. It is concluded that effects of the photosynthesis inhibiting herbicide on aquatic macrophytes, algae and algae encapsulated in alginate beads can be conveniently evaluated by measuring photosystem II electron transport by means of chlorophyll fluorescence. This method can be used as a rapid and non-destructive technique in aquatic ecological research. This revised version was published online in August 2006 with corrections to the Cover Date.     

Ecotoxicological threshold levels of a mixture of herbicides (atrazine, diuron and metolachlor) in freshwater microcosms

Twelve indoor, plankton-dominated, freshwater microcosms (600 l) were used to study the effect of a mixture of herbicides on structural and functional aspects of these ecosystems. The EC50, 72 h values of the most susceptible standard test alga Selenastrum capricornutum (EC50, atrazine=54 μg l−1, EC50, diuron=15 μg l−1, EC50, metolachlor=56 μg l−1) were used as a starting point for the dosage applied in the microcosms (dosages: 0, 0.01, 0.03, 0.1, 0.3, 1× EC50). The microcosms were exposed to chronic levels for 28 days and subsequently monitored for 4 more weeks. The following effects were observed: (1) direct effects became apparent from an initial drop in photosynthesis efficiency, pH and oxygen concentration and a decrease in the abundance of several phytoplankton taxa at the 0.3 × EC50 treatment level and higher. (2) Fourteen days post application an increase in the abundance of several phytoplankton taxa (Chlamydomonas sp. and Stephanodiscus/Cyclotella) was observed; oxygen concentrations recovered while alkalinity, conductivity and total inorganic nitrogen were elevated. (3) Effects on fauna were minor. Daphnia galeata showed a decreasing trend and the cyclopoid copepods an increasing trend at the end of the experiment. Multivariate statistical analyses demonstrated no effects of any treatment level on the zooplankton community. Effects were reported for the phytoplankton community at dose levels of 0.3 × EC50 and higher. On species level the most sensitive taxon was Chlorophyceae coccales. For this taxon a NOEC at the dose level of 0.01 × EC50 was calculated. This effect however was relatively small in magnitude and merely based on an increase in numbers in the control and lowest treated microcosms rather than a decrease in numbers in all other treatments. The standards based on algal toxicity data, as adopted by the Uniform Principles, consist of a safety factors of 0.1 to be multiplied with the EC50. The NOEC of coccales was lower than 0.1 × EC50. All other observed variables in this aquatic ecosystem were sufficiently protected against the mixture of herbicides by the safety factor as proposed in the Uniform Principles. This revised version was published online in August 2006 with corrections to the Cover Date.