UreG, a chaperone in the urease assembly process, is an intrinsically unstructured GTPase that specifically binds Zn2+

Bacillus pasteurii UreG, a chaperone involved in the urease active site assembly, was overexpressed in Escherichia coli BL21(DE3) and purified to homogeneity. The identity of the recombinant protein was confirmed by SDS-PAGE, protein sequencing, and mass spectrometry. A combination of size exclusion chromatography and multiangle and dynamic laser light scattering established that BpUreG is present in solution as a dimer. Analysis of circular dichroism spectra indicated that the protein contains large portions of helices (15%) and strands (29%), whereas NMR spectroscopy indicated the presence of conformational fluxionality of the protein backbone in solution. BpUreG catalyzes the hydrolysis of GTP with a kcat=0.04 min(-1), confirming a role for this class of proteins in coupling energy requirements and nickel incorporation into the urease active site. BpUreG binds two Zn2+ ions per dimer, with a KD=42 +/- 3 microm, and has a 10-fold lower affinity for Ni2+. A structural model for BpUreG was calculated by using threading algorithms. The protein, in the fully folded state, features the typical structural architecture of GTPases, with an open beta-barrel surrounded by alpha-helices and a P-loop at the N terminus. The protein dynamic behavior observed in solution is critically discussed relative to the structural model, using algorithms for disorder predictions. The results suggest that UreG proteins belong to the class of intrinsically unstructured proteins that need the interaction with cofactors or other protein partners to perform their function. It is also proposed that metal ions such as Zn2+ could have important structural roles in the urease activation process.     

Nickel trafficking: insights into the fold and function of UreE, a urease metallochaperone

UreE is a metallo-chaperone assisting the incorporation of two adjacent Ni(2+) ions in the active site of urease. This study describes an attempt to distill general information on this protein using a computational post-genomic approach for the understanding of the structural details of the molecular function of UreE in nickel trafficking. The two crystal structures recently determined for UreE from Bacillus pasteurii (BpUreE) and Klebsiella aerogenes (KaUreE) were comparatively analyzed. This analysis provided insights into the protein structural and conformational features. A structural database of UreE proteins from a large number of different genomes was built using homology modeling. All available sequences of UreE were retrieved from protein and cDNA databases, and their structures were modeled on the crystal structures of BpUreE and KaUreE. A self-consistent iterative protocol was devised for multiple sequence alignment optimization involving secondary structure prediction and evaluation of the energy features of the obtained modeled structures. The quality of all models was tested using standard assessment procedures. The final optimized structure-based multiple alignment and the derived model structures provided insightful information on the evolutionary conservation of key residues in the protein sequence and surface patches presumably involved in protein recognition during the urease active site assembly.     

Identification of camelid specific residues in mitochondrial ATP synthase subunits

ATP synthase is an enzyme involved in oxidative phosphorylation from prokaryotic to eukaryotic cells. In mammals it comprises at least 16 subunits from which the mitochondrial encoded ATP6 and ATP8 are essential. Mitochondrial genes variations have been suggested to allow rapid human and animal adaptation to new climates and dietary conditions (Mishmar et al. 2003). Camelidae taxa are uniquely adapted to extremely hot and dry climates of African-Asian territories and to cold and hypoxic environments of the South American Andean region. We sequenced and analyzed ATP6 and ATP8 genes in all camelid species. Based on the available structural data and evolutionary conservation of the deduced proteins we identified features proper of the group. In Old World camels the ATP8, important in the assembly of the F0 complex, showed a number of positively charged residues higher than in the other aligned species. In ATP6 we found the camelid specific substitutions Q47H and I106V that occur in sites highly conserved in other species. We speculate that these changes may have functional importance.     

In vitro evaluation of fresh sperm quality in tomcats: A comparison of two collection techniques

The collection of semen from tomcats by urethral catheterization (CT) after medetomidine administration offers a novel and easy approach to obtain good quality sperm for in vitro fertilization. This study was designed to compare the sperm quality parameters and in vitro fertilizing capacity of CT spermatozoa with those of spermatozoa retrieved after epididymal slicing (EP). Semen was collected in seventeen adult cats by urethral catheterization, after which the cat was orchiectomized. Motility, morphology, plasma membrane integrity, acrosomal status, and in vitro fertilizing capacity of both fresh CT and EP samples were evaluated. The results showed that both total and progressive motility, as well as the percentage of normal spermatozoa, were higher for EP sperm than for CT sperm (P < 0.01). Epididymal sperm had a lower percentage of spermatozoa with an intact acrosome (P < 0.01), while CT sperm contained more spermatozoa with tail abnormalities (P < 0.01). Other morphological parameters, as well as plasma membrane integrity, did not differ (P > 0.05) between CT and EP sperm. Nevertheless, no difference (P > 0.05) in in vitro fertilizing capacity between spermatozoa collected by means of the two different methods was found. In conclusion, semen collection by means of urethral catheterization after medetomidine administration yields fertilization results similar to epididymal slicing, despite the fact that several sperm variables were different. Since this novel catheterization technique is repeatable, is easy to perform and facilitates semen preparation protocols, it may be preferable for routine IVF experiments with fresh spermatozoa.     

Correlation between the age of the conceptus and various ultrasonographic measurements during the first 30 days of pregnancy in domestic cats (Felis catus)

We ultrasonographically evaluated the prenatal development in cats, from the early phases to Day 30 of pregnancy, subjecting a group of pregnant cats (n = 12) to a daily ultrasonographic exam. The ultrasonographic images allowed us to measure the minor diameter of the gestational sac and the crown-rump length of the embryo/fetus. Ten subjects underwent ovariohysterectomy at specific intervals during the pregnancy, with the aim of comparing the ultrasonographic data with real data; only two subjects brought their pregnancy to term. The earliest ultrasonographic observation of the gestational sac was on Day 10 after mating, while the embryo could be measured only beginning with Day 18. This study allowed to gather useful new data in order to clinically monitor the normal course of pregnancy in cats and to date the gestational age.     

Peri-implant bone remodeling after total hip replacement combined with systemic alendronate treatment: a finite element analysis

In order to decrease the peri-implant bone loss during the life-time of the implant, oral use of anti-osteoporosis drugs (like bisphosphonates) has been suggested. In this study, bone remodeling parameters identified from clinical trials of alendronate were used to simulate the effect of those drugs used after total hip arthroplasty on the peri-implant bone density. Results of the simulation show that the oral administrated drugs increase bone density around the implant and decreases, at the same time, the micromovements between the implant and the surrounding bone tissue. Incorporation of drug effect in numerical studies of bone remodeling is a promising tool especially to predetermine safe bisphosphonate doses that could be used with orthopedic implants.