Effect of 12-months testosterone replacement therapy on bone mineral density and markers of bone turnover in testicular cancer survivors - results from a randomized double-blind trial.

BACKGROUND: Testicular cancer survivors (TCS) are at risk of Leydig cell insufficiency, which is a condition characterized by elevated luteinising hormone (LH) in combination with low levels of testosterone. It has been suggested that this condition is associated with impaired metabolic profile and low bone mineral density (BMD). The primary aim of the randomized double-blind trial NCT02991209 was to evaluate metabolic profile after 12-months testosterone replacement therapy (TRT) in TCS with mild Leydig cell insufficiency. Here we present the secondary outcomes of changes in BMD and markers of bone turnover. METHODOLOGY: In total, 69 TCS with mild Leydig cell insufficiency were randomized 1:1 to 12 months TRT (n = 35) (Tostran, gel, 2%, applied transdermally, with a maximum daily dose of 40 mg) or placebo (n = 34). BMD and markers of bone turnover were evaluated at baseline, after 6- and 12-months TRT, and 3-months post-treatment. Linear mixed effects models were used to analyse changes in BMD, N-terminal propeptide of type 1 procollagen (P1NP) and C-terminal telopeptide of type I collagen (CTX). RESULTS: After 12 months treatment, TRT was not associated with a statistically significant difference in BMD compared to placebo; total body BMD: 0.01 g/cm2 (95% confidence interval (CI): -0.01 - 0.02), BMD of the lumbar spine: 0.01 g/cm2, (95% CI: -0.01-0.03), BMD of the left femoral neck: 0.00, (95% CI: -0.01-0.02). TRT was associated with a small but statistically significant increase in P1NP: 11.65 µg/L (95% CI: 3.96, 19.35), while there was no difference in CTX. CONCLUSION: 12 months of TRT did not change BMD, while there was as small and clinically irrelevant increase in P1NP compared to placebo in TCS with mild Leydig cell insufficiency. The findings need validation in a larger cohort.

Homology modeling of Na,K-ATPase: a putative third sodium binding site suggests a relay mechanism compatible with the electrogenic profile of Na+ translocation.

Identification of the third Na(+) binding site would be crucial in interpretation of the electrophysiological behavior of Na,K-ATPase. To address this question a three-dimensional homology model of Na,K-ATPase was built from the known crystallographic structure of Ca-ATPase (1EUL). Phe760, which is conserved in virtually all Ca-ATPases, is replaced by Ser768 in Na,K-ATPase, resulting in a small cavity between M4, M5, and M6. A partially hydrated Na(+) ion can be bound at this third site on the cytoplasmic side of cation binding sites 1 and 2. This leads to the proposal that the conductance of the "third Na(+)" ion across approximately 70% of the membrane dielectric may be achieved by adding up the passage of one Na(+) ion from the described cytoplasmic cavity to cation site 1 and the further conductance of the previously bound Na(+) ion from cation site 1 to the extracellular phase. This relay mechanism may therefore be compatible with the electrogenic profile of Na(+) translocation.

Trafficking of Na,K-ATPase fused to enhanced green fluorescent protein is mediated by protein kinase A or C.

Fusion of enhanced green fluorescent protein (EGFP) to the C-terminal of rat Na,K-ATPase a1-subunit is introduced as a novel procedure for visualizing trafficking of Na,K-pumps in living COS-1 renal cells in response to PKA or PKC stimulation. Stable, functional expression of the fluorescent chimera (Na,K-EGFP) was achieved in COS-1 cells using combined puromycin and ouabain selection procedures. Na,K-pump activities were unchanged after fusion with EGFP, both in basal and regulated states. In confocal laser scanning and fluorescence microscopes, the Na,K-EGFP chimera was distributed mainly along the plasma membrane of COS cells. In unstimulated COS cells, Na,K-EGFP was also present in lysosomes and in vesicles en route from the endoplasmic reticulum to the plasma membrane, but it was almost absent from recycling endosomes labelled with fluorescent transferrin. After activation of protein kinase A or C, the density of co-localizing Na,K-EGFP and transferrin vesicles was increased 3-4-fold, while the ouabain-sensitive 86Rb uptake was reduced by 22%. Simultaneous activation of PKA and PKC had additive effects with a 6-fold increase of co-localization and a 38% reduction of 86Rb uptake. Responses of similar magnitude were seen after inhibition of protein phosphatase by okadaic acid. Reduction of the amount of Na,K-ATPase in surface plasma membranes through internalization in recycling endosomes may thus in part explain a decrease in Na,K-pump activity following protein kinase activation or protein phosphatase inhibition.

Aspects of gene structure and functional regulation of the isozymes of Na,K-ATPase.

Gaps in our understanding of the complex regulated expression of isozymes of Na,K-ATPase and the diverse systems for posttranslational modification and short term regulation of active Na,K-transport in animals and humans are the main problems in comprehensive Na,K-pump physiology. In mammalian genomes, the genes of four alpha-subunit and at least three beta-subunit isoforms of Na,K-ATPase are identified and two gamma-subunits are expressed in kidney. The isoforms combine in a number of Na,K-ATPase isozymes that are expressed in a tissue and cell specific manner. Models of the molecular mechanism of regulation of these isozymes have become more reliable due to progress in understanding the three-dimensional protein structure and conformational transitions mediating transfer of energy from the P-domain to intramembrane Na+ and K+ binding sites.

Structure-function relationships of Na(+), K(+), ATP, or Mg(2+) binding and energy transduction in Na,K-ATPase.

The focus of this article is on progress in establishing structure-function relationships through site-directed mutagenesis and direct binding assay of Tl(+), Rb(+), K(+), Na(+), Mg(2+) or free ATP at equilibrium in Na,K-ATPase. Direct binding may identify residues coordinating cations in the E(2)[2K] or E(1)P[3Na] forms of the ping-pong reaction sequence and allow estimates of their contributions to the change of Gibbs free energy of binding. This is required to understand the molecular basis for the pronounced Na/K selectivity at the cytoplasmic and extracellular surfaces. Intramembrane Glu(327) in transmembrane segment M4, Glu(779) in M5, Asp(804) and Asp(808) in M6 are essential for tight binding of K(+) and Na(+). Asn(324) and Glu(327) in M4, Thr(774), Asn(776), and Glu(779) in 771-YTLTSNIPEITP of M5 contribute to Na(+)/K(+) selectivity. Free ATP binding identifies Arg(544) as essential for high affinity binding of ATP or ADP. In the 708-TGDGVND segment, mutations of Asp(710) or Asn(713) do not interfere with free ATP binding. Asp(710) is essential and Asn(713) is important for coordination of Mg(2+) in the E(1)P[3Na] complex, but they do not contribute to Mg(2+) binding in the E(2)P-ouabain complex. Transition to the E(2)P form involves a shift of Mg(2+) coordination away from Asp(710) and Asn(713) and the two residues become more important for hydrolysis of the acyl phosphate bond at Asp(369).

Role of conserved TGDGVND-loop in Mg2+ binding, phosphorylation, and energy transfer in Na,K-ATPase.

In the P-domain, the 369-DKTGTLT and the 709-GDGVNDSPALKK segment are highly conserved during evolution of P-type E1-E2-ATPase pumps irrespective of their cation specificities. The focus of this article is on evaluation of the role of the amino acid residues in the P domain of the alpha subunit of Na,K-ATPase for the E1P[3Na]--> E2P[2Na] conversion, the K+-activated dephosphorylation, and the transmission of these changes to and from the cation binding sites. Mutations of residues in the TGDGVND loop show that Asp710 is essential, and Asn713 is important, for Mg2+ binding and formation of the high-energy MgE1P[3Na] intermediate. In contrast Asp710 and Asp713 do not contribute to Mg2+ binding in the E2P-ouabain complex. Transition to E2P thus involves a shift of Mg2+ coordination away from Asp710 and Asn713 and the two residues become more important for K+-activated hydrolysis of the acyl phosphate bond at Asp369. Transmission of structural changes between the P-domain and cation sites in the membrane domain is evaluated in light of the protein structure, and the information from proteolytic or metal-catalyzed cleavage and mutagenesis studies.

Using inactivated microbial biomass as fertilizer: the fate of antibiotic resistance genes in the environment.

The waste product produced by Novo Nordisk A/S from microbial fermentations is used as agricultural fertilizer in Denmark (NovoGro) after being treated by heat and chemicals to destroy the microorganisms. The fertilizer contains DNA fragments from the genetically modified microorganisms used in industrial production. This DNA contains genes coding for the desired industrial products as well as genes used as genetic selection markers during production strain development. The antibiotic resistance markers used as genetic selection markers are chloramphenicol (Cm), kanamycin (Km) and ampicillin (Ap). The aim of the present study was to examine whether DNA and intact genes were present in NovoGro and whether horizontal transfer of DNA isolated from inactivated production strains occurred either in the laboratory or in the fields treated with NovoGro. DNA isolated from NovoGro was analysed by PCR and intact genes coding for a protease and chloramphenicol resistance were amplified. This isolated DNA was used for in vitro experiments including electroporation and transformation but no transfer of DNA to Escherichia coli or Bacillus subtilis was observed. The antibiotic resistance profile of the indigenous bacterial population in the fields treated with NovoGro compared with fields treated with inorganic fertilizers showed no differences. In addition, DNA isolated directly from the fields treated with NovoGro for up to 7 years was analysed by PCR and no specific production gene constructs could be detected.

Importance of conserved alpha -subunit segment 709GDGVND for Mg2+ binding, phosphorylation, and energy transduction in Na,K-ATPase.

The segment (708)TGDGVNDSPALKK(720) in the alpha-subunit P domain of Na,K-ATPase is highly conserved among cation pumps, but little is known about its role in binding of Mg(2+) or ATP and energy transduction. Here, 11 mutations of polar residues are expressed at reduced temperature in yeast with preserved capacities for high affinity binding of ouabain and ATP, whereas the Thr(708) --> Ser mutation and alterations of Asp(714) abolish all catalytic reactions. In mutations of Asp(710) and Asn(713), ATP affinity is preserved or increased, whereas Na,K-ATPase activity is severely reduced. Assay of phosphorylation from ATP in the presence of oligomycin shows that Asp(710) contributes to coordination of Mg(2+) during transfer of gamma-phosphate to Asp(369) in the high energy Mg.E(1)P[3Na] intermediate and that Asn(713) is involved in these processes. In contrast, Asp(710) and Asp(713) do not contribute to Mg(2+) binding in the E(2)P.ouabain complex. Transition to E(2)P thus involves a shift of Mg(2+) coordination away from Asp(710) and Asn(713), and the two residues become more important for hydrolysis of the acyl phosphate bond at Asp(369). The Asp(710) --> Ala mutation blocks interaction with vanadate, whereas Asn(713) --> Ala interferes with phosphorylation from P(i) of the E(2).ouabain complex, showing that the GDGVND segment is required for stabilization of the transition state and for the phosphorylation reaction. The Asp(710) --> Ala mutation also interferes with transmission of structural changes to the ouabain site and reduces the affinity for binding of Tl(+) 2- to 3-fold, suggesting a role in transmission of K(+) stimulation of phospho-enzyme hydrolysis from transmembrane segment 5 to the P domain.

Cloning and sequencing of an alkaline protease gene from Bacillus lentus and amplification of the gene on the B. lentus chromosome by an improved technique.

A gene encoding an alkaline protease was cloned from an alkalophilic bacillus, and its nucleotide sequence was determined. The cloned gene was used to increase the copy number of the protease gene on the chromosome by an improved gene amplification technique.

Catalysis and specificity in enzymatic glycoside hydrolysis: a 2,5B conformation for the glycosyl-enzyme intermediate revealed by the structure of the Bacillus agaradhaerens family 11 xylanase.

BACKGROUND: The enzymatic hydrolysis of glycosides involves the formation and subsequent breakdown of a covalent glycosyl-enzyme intermediate via oxocarbenium-ion-like transition states. The covalent intermediate may be trapped on-enzyme using 2-fluoro-substituted glycosides, which provide details of the intermediate conformation and noncovalent interactions between enzyme and oligosaccharide. Xylanases are important in industrial applications - in the pulp and paper industry, pretreating wood with xylanases decreases the amount of chlorine-containing chemicals used. Xylanases are structurally similar to cellulases but differ in their specificity for xylose-based, versus glucose-based, substrates. RESULTS: The structure of the family 11 xylanase, Xyl11, from Bacillus agaradhaerens has been solved using X-ray crystallography in both native and xylobiosyl-enzyme intermediate forms at 1.78 A and 2.0 A resolution, respectively. The covalent glycosyl-enzyme intermediate has been trapped using a 2-fluoro-2-deoxy substrate with a good leaving group. Unlike covalent intermediate structures for glycoside hydrolases from other families, the covalent glycosyl-enzyme intermediate in family 11 adopts an unusual 2,5B conformation. CONCLUSIONS: The 2,5B conformation found for the alpha-linked xylobiosyl-enzyme intermediate of Xyl11, unlike the 4C1 chair conformation observed for other systems, is consistent with the stereochemical constraints required of the oxocarbenium-ion-like transition state. Comparison of the Xyl11 covalent glycosyl-enzyme intermediate with the equivalent structure for the related family 12 endoglucanase, CelB, from Streptomyces lividans reveals the likely determinants for substrate specificity in this clan of glycoside hydrolases.

Pullulanase type I from Fervidobacterium pennavorans Ven5: cloning, sequencing, and expression of the gene and biochemical characterization of the recombinant enzyme.

The gene encoding the type I pullulanase from the extremely thermophilic anaerobic bacterium Fervidobacterium pennavorans Ven5 was cloned and sequenced in Escherichia coli. The pulA gene from F. pennavorans Ven5 had 50.1% pairwise amino acid identity with pulA from the anaerobic hyperthermophile Thermotoga maritima and contained the four regions conserved among all amylolytic enzymes. The pullulanase gene (pulA) encodes a protein of 849 amino acids with a 28-residue signal peptide. The pulA gene was subcloned without its signal sequence and overexpressed in E. coli under the control of the trc promoter. This clone, E. coli FD748, produced two proteins (93 and 83 kDa) with pullulanase activity. A second start site, identified 118 amino acids downstream from the ATG start site, with a Shine-Dalgarno-like sequence (GGAGG) and TTG translation initiation codon was mutated to produce only the 93-kDa protein. The recombinant purified pullulanases (rPulAs) were optimally active at pH 6 and 80 degrees C and had a half-life of 2 h at 80 degrees C. The rPulAs hydrolyzed alpha-1,6 glycosidic linkages of pullulan, starch, amylopectin, glycogen, alpha-beta-limited dextrin. Interestingly, amylose, which contains only alpha-1,4 glycosidic linkages, was not hydrolyzed by rPulAs. According to these results, the enzyme is classified as a debranching enzyme, pullulanase type I. The extraordinary high substrate specificity of rPulA together with its thermal stability makes this enzyme a good candidate for biotechnological applications in the starch-processing industry.

Structure-function relationships based on ATP binding and cation occlusion at equilibrium in Na,K-ATPase.

This work evaluates the results of measurements of equilibrium binding of ATP and cations in lethal or partially active mutations of Na,K-ATPase that were expressed at high yield in yeast cells. ATP binding studies allowed estimation of the expense in free energy required to position the gamma-phosphate in proximity of the carboxylate groups of the phosphorylated residue Asp369 and the role of this residue in governing long range E1-E2 transitions. An arginine residue (Arg546) appearing to be involved in ATP binding has been identified. Wild type yeast enzyme was capable of occluding two T1(+)-ions per ouabain binding site or alpha 1 beta 1 unit with high apparent affinity (Kd(T1+) = 7 +/- 2 microM), like the purified Na,K-ATPase from pig kidney. The substitutions to Glu327(Gln,Asp), Asp804(Asn,Glu), Asp808(Asn,Glu) and Glu779(Asp) completely abolished occlusion or severely reduced the affinity for T1+ ions. The substitution of Glu779 for Gln reduced the occlusion capacity to one T1+ ion per alpha 1 beta 1 unit with a 3-fold decrease of the apparent affinity for the ion (Kd(T1+) = 24 +/- 8 mM). These carboxylate groups in transmembrane segments 4, 5, and 6 therefore appear to be essential for high affinity occlusion of K(+)-ions.

Structure-function relationships of E1-E2 transitions and cation binding in Na,K-pump protein.

Fully active Na,K-ATPase and lethal mutations can be expressed in yeast cells in yields allowing for equilibrium ATP binding, occlusion of T1+, K+ displacement of ATP, and Na(+)-dependent phosphorylation with determinations of affinity constants for binding and constants for the conformational equilibria. Removal of the charge and hydrophobic substitution of the phosphorylated residue (Asp369Ala) reveals an intrinsic high affinity for ATP binding (Kd 2.8 vs. 100 nM for wild type) and causes a shift of conformational equilibrium towards the E2 form. Substitution of Glu327, Glu779, Asp804 or Asp808 in transmembrane segments 4, 5, and 6 shows that each of these residues are essential for high-affinity occlusion of K+ and for binding of Na+. Substitution of other residues in segment 5 shows that the carboxamide group of Asn776 is important for binding of both K+ and Na+. Differential effects of the relevant mutations identify Thr774 as specific determinant of Na+ binding in the E1P[3Na] form, whereas Ser775 is a specific participant of high-affinity binding of the E2[2K] form, suggesting that these residues engage in formation of a molecular Na+/K+ switch. The position of the switch may be controlled by rotating or tilting the helix during the E1-E2 transition.

Importance of intramembrane carboxylic acids for occlusion of K+ ions at equilibrium in renal Na,K-ATPase.

Site-directed mutagenesis and assay of Rb+ and Tl+ occlusion in recombinant Na,K-ATPase from yeast were combined to establish structure-function relationships of amino acid side chains involved in high-affinity occlusion of K+ in the E2[2K] form. The wild-type yeast enzyme was capable of occluding 2 Rb+ or Tl+ ions/ouabain binding site or alpha 1 beta 1 unit with high apparent affinity (Kd(Tl+) = 7 +/- 2 microM), like the purified Na,K-ATPase from pig kidney. Mutations of Glu327(Gln,Asp), Asp804(Asn, Glu), Asp808(Asn, Glu) and Glu779(Asp) abolished high-affinity occlusion of Rb+ or Tl+ ions. The substitution of Glu779 for Gln reduced the occlusion capacity to 1 Tl+ ion/alpha 1 beta 1-unit with a 3-fold decrease of the apparent affinity for the ion (Kd(Tl+) = 24 +/- 8 microM). These effects on occlusion were closely correlated to effects of the mutations on K0.5(K+) for K+ displacement of ATP binding. Each of the four carboxylate residues Glu327, Glu779, and Asp804 or Asp808 in transmembrane segments 4, 5, and 6 is therefore essential for high-affinity occlusion of K+ in the E2[2K] form. These residues either may engage directly in cation coordination or they may be important for formation or stability of the occlusion cavity.

Contribution to Tl+, K+, and Na+ binding of Asn776, Ser775, Thr774, Thr772, and Tyr771 in cytoplasmic part of fifth transmembrane segment in alpha-subunit of renal Na,K-ATPase.

The sequence Y771TLTSNIPEIT781P in the fifth transmembrane segment of the alpha-subunit of Na,K-ATPase is unique among cation pump proteins. Here, in search of the molecular basis for Na,K specificity, alanine and conservative substitutions were directed to six oxygen-carrying residues in this segment. The contribution of the residues to cation binding was estimated from direct binding of Tl+ [Nielsen, et al. (1998) Biochemistry 37, 1961-1968], K+ displacement of ATP binding at equilibrium, and Na+-dependent phosphorylation from ATP in the presence of oligomycin. As an intrinsic control, substitution of Thr781 had no effect on Tl+(K+) or Na+ binding. There are several novel observations from this work. First, the carboxamide group of Asn776 is equally important for binding Tl+(K+) or Na+, whereas a shift of the position of the carboxamide of Asn776 (Asn776Gln) causes a large depression of Na+ binding without affecting the binding of Tl+(K+). Second, Thr774 is important for Na+ selectivity because removal of the hydroxyl group reduces the binding of Na+ with no effect on binding of Tl+(K+). Removal of the methyl groups of Thr774 or Thr772 reduces binding of both Tl+(K+) and Na+, whereas the hydroxyl group of Thr772 does not contribute to cation binding. Furthermore, the hydroxyl groups of Ser775 and Tyr771 are important for binding both Tl+(K+) and Na+. The data suggest that rotating or tilting of the cytoplasmic part of the fifth transmembrane segment may adapt distances between coordinating groups and contribute to the distinctive Na+/K+ selectivity of the pump.

Identification of Asp804 and Asp808 as Na+ and K+ coordinating residues in alpha-subunit of renal Na,K-ATPase.

Mutations to Asp804 and Asp808 in the alpha-subunit almost abolish Na,K-ATPase activity, but high-affinity binding of [3H]ATP or [3H]ouabain at equilibrium and E1-E2 transitions are preserved. Titration of K+-ion displacement of [3H]ATP or [3H]ouabain shows that the mutations interfere with occlusion of K+ in the E2[2K] conformation. Reduced phosphorylation levels or affinities for Na+ in presence of oligomycin indicate that Asp804 and Asp808 also contribute to coordination of Na+ in the E1P[3Na] form. Demonstration of alternate interactions of Na+ or K+ with Asp804 and Asp808 support the notion of cation binding in a ping-pong sequence in catalytic models of Na,K-pumping.