Localization of polyamine enhancement of protein synthesis to subcellular components of Escherichia coli and Pseudomonas sp. strain Kim.

At 5 mM Mg2+, spermidine stimulation of polyphenylalanine synthesis by cell-free extracts of Escherichia coli was found to be about 30 times greater than that by extracts of Pseudomonas sp. strain Kim, a unique organism which lacks detectable levels of spermidine. By means of reconstitution experiments, the target of spermidine stimulation was localized to the protein fraction of the highspeed supernatant component (S-100) of E. coli and was absent from, or deficient in, the S-100 fraction of Pseudomonas sp. strain Kim. The spermidine stimulation did not appear to be due to the presence in the E. coli S-100 fraction of ribosomal protein S1, elongation factors, or E. coli aminoacyl-tRNA synthetases. The failure to observe spermidine stimulation by the Pseudomonas sp. strain Kim S-100 fraction was also not due to a spermidine-enhanced polyuridylic acid degradation. The synthesis of polyphenylalanine by Pseudomonas sp. strain Kim extracts was stimulated by putrescine and by S-(+)-2-hydroxyputrescine to a greater degree than was synthesis by E. coli extracts. The enhancement by putrescine and by S-(+)-2-hydroxyputrescine with Pseudomonas sp. strain Kim extracts was found to be due to effects on its ribosomes.     

Newly discovered polyamine, 2-hydroxyspermidine, in Pseudomonas acidovorans.

A previously unknown hydroxylated polyamine has been recovered from Pseudomonas acidovorans 29. It has been identified as 2-hydroxyspermidine, N4-(3-aminopropyl)-1,4-diaminobutane-2-ol, by its chromatographic behavior, electrophoretic mobility, and reaction with metaperiodate. It can be synthesized enzymatically from 2-hydroxyputrescine by cell-free preparations from Escherichia coli or P. acidovorans 29 which contain propylamine transferase. It is interesting to note that the naturally occurring compound is the 2-hydroxyspermidine and not the 3-hydroxyspermidine, N1-(3-aminopropyl)-1,4-diaminobutane-2-ol, indicating that the propylamine transferase reacts preferentially with the amine distal to the hydroxyl group. A mixture of 2- and 3-hydroxyspermidines and hydroxyspermine was synthesized by reacting acrylonitrile with 2-hydroxyspermidine and catalytic reduction of the products with hydrogen. N-(gamma-aminopropyl)-beta-alanine, used to help identify the hydroxyspermidines, was synthesized from N-(3-aminopropyl)-3-aminopropanenitrile by hydrolysis with 10% NaOH.     

Developmental changes in mitochondria during the transition into lactation in the mouse mammary gland. II. Membrane marker enzymes and membrane ultrastructure

The activity of cytochrome oxidase (an inner mitochondrial membrane marker) in mouse mammary gland homogenates was found to increase five- to sixfold from late pregnancy to day 8 of lactation, while that of monoamine oxidase (an outer membrane marker) increased only about 25%. The specific activity of cytochrome oxidase in the isolated mitochondria decreased slightly over the same period while the specific activity of monoamine oxidase decreased fivefold. This reflects the fact that both cytochrome oxidase and mitochondrial protein are increasing at a much greater rate than is monoamine oxidase activity. Mixing experiments preclude the possibility that the release or removal of an inhibitor or stimulator produces the changes in enzymatic activity. The cytochrome oxidase to monoamine oxidase ratio was followed throughout the pregnancy-lactation cycle in total mammary homogenates, isolated mammary parenchymal cells, and isolated mammary mitochondria. In each preparation the pattern was the same with little change in the ratio until late pregnancy; and then a three- to fourfold increase occurred and the values reached a maximum by day 8 of lactation. These experiments were interpreted as demonstrating that the observed enzymatic changes are reflective of alterations in the mitochondria of the mammary parenchymal cell population. Electron micrographs of mid-pregnant and mid-lactating mammary parenchymal cells in situ were prepared, and distinct changes in the mitochondrial morphology noted. The most significant and obvious change is the large increase in the number of inner membrane cristae and an increase in matrix density in the lactating gland cell. Therefore, both enzymatic and morphological studies support the concept of an expansion of the mitochondrial inner membrane during presecretory differentiation in the mouse mammary parenchymal cell.     

Oxygen binding by alpha(Fe2+)2beta(Ni2+)2 hemoglobin crystals

Oxygen binding by hemoglobin fixed in the T state either by crystallization or by encapsulation in silica gels is apparently noncooperative. However, cooperativity might be masked by different oxygen affinities of alpha and beta subunits. Metal hybrid hemoglobins, where the noniron metal does not bind oxygen, provide the opportunity to determine the oxygen affinities of alpha and beta hemes separately. Previous studies have characterized the oxygen binding by alpha(Ni2+)2beta(Fe2+)2 crystals. Here, we have determined the three-dimensional (3D) structure and oxygen binding of alpha(Fe2+)2beta(Ni2+)2 crystals grown from polyethylene glycol solutions. Polarized absorption spectra were recorded at different oxygen pressures with light polarized parallel either to the b or c crystal axis by single crystal microspectrophotometry. The oxygen pressures at 50% saturation (p50s) are 95 +/- 3 and 87 +/- 4 Torr along the b and c crystal axes, respectively, and the corresponding Hill coefficients are 0.96 +/- 0.06 and 0.90 +/- 0.03. Analysis of the binding curves, taking into account the different projections of the alpha hemes along the optical directions, indicates that the oxygen affinity of alpha1 hemes is 1.3-fold lower than alpha2 hemes. Inspection of the 3D structure suggests that this inequivalence may arise from packing interactions of the Hb tetramer within the monoclinic crystal lattice. A similar inequivalence was found for the beta subunits of alpha(Ni2+)2beta(Fe2+)2 crystals. The average oxygen affinity of the alpha subunits (p50 = 91 Torr) is about 1.2-fold higher than the beta subunits (p50 = 110 Torr). In the absence of cooperativity, this heterogeneity yields an oxygen binding curve of Hb A with a Hill coefficient of 0.999. Since the binding curves of Hb A crystals exhibit a Hill coefficient very close to unity, these findings indicate that oxygen binding by T-state hemoglobin is noncooperative, in keeping with the Monod, Wyman, and Changeux model.     

Toward a unified model of the action of CLP/HSP100 chaperones in chloroplasts

In chloroplasts, Hsp70 and Hsp100 chaperones have been long suspected to be the motors that provide the necessary energy for the import of precursor proteins destined to the organelle. The chaperones associate with the import translocon and meet the transit peptides as they emerge through the channel. After decades of active research, recent findings demonstrated that Hsp100 chaperones recognize transit peptides both in vitro and in vivo. Moreover, Hsp70 also plays a part in precursor import. The updated model of protein translocation into chloroplasts now presents new questions about the role of the chaperones in the process.     

The X-ray three-dimensional structure of avidin

Avidin is a basic, highly stable, homotetrameric protein, isolated from bird egg-white, binding up to four molecules of D-biotin with extremely high affinity (Kd approximately 10(-15) M). The protein has been the object of different crystallographic investigations. In all the crystal structures, the four avidin subunits display almost exact 222 symmetry. Each avidin chain (128 amino acids) is arranged in a eight-stranded antiparallel beta-barrel, whose inner region defines the D-biotin binding site. The molecular bases of D-biotin affinity can be recognised in a fairly rigid binding site, which is sterically complementary to the shape and polarity of the incoming vitamin, and is readily accessible in the apoprotein structure. Avidin displays remarkable structural and functional relationships to the acidic protein sretpavidin, isolated from Streptomyces avidinii.     

Spermidine synthesis by Pseudomonas sp. strain Kim, previously reported to lack this polyamine.

Pseudomonas sp. strain Kim has previously been reported to be the only known naturally occurring organism lacking spermidine. We now show that it synthesizes this polyamine. The apparent lack of intracellular levels of spermidine results from an efficient conversion of spermidine to putrescine and hydroxyputrescine.     

Cardiovascular and Renal Outcomes of Renin–Angiotensin System Blockade in Adult Patients with Diabetes Mellitus: A Systematic Review with Network Meta-Analyses

Background

Medications aimed at inhibiting the renin–angiotensin system (RAS) have been used extensively for preventing cardiovascular and renal complications in patients with diabetes, but data that compare their clinical effectiveness are limited. We aimed to compare the effects of classes of RAS blockers on cardiovascular and renal outcomes in adults with diabetes.

Methods and Findings

Eligible trials were identified by electronic searches in PubMed/MEDLINE and the Cochrane Database of Systematic Reviews (1 January 2004 to 17 July 2014). Interventions of interest were angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), and direct renin (DR) inhibitors. The primary endpoints were cardiovascular mortality, myocardial infarction, and stroke—singly and as a composite endpoint, major cardiovascular outcome—and end-stage renal disease [ESRD], doubling of serum creatinine, and all-cause mortality—singly and as a composite endpoint, progression of renal disease. Secondary endpoints were angina pectoris and hospitalization for heart failure. In all, 71 trials (103,120 participants), with a total of 14 different regimens, were pooled using network meta-analyses. When compared with ACE inhibitor, no other RAS blocker used in monotherapy and/or combination was associated with a significant reduction in major cardiovascular outcomes: ARB (odds ratio [OR] 1.02; 95% credible interval [CrI] 0.90–1.18), ACE inhibitor plus ARB (0.97; 95% CrI 0.79–1.19), DR inhibitor plus ACE inhibitor (1.32; 95% CrI 0.96–1.81), and DR inhibitor plus ARB (1.00; 95% CrI 0.73–1.38). For the risk of progression of renal disease, no significant differences were detected between ACE inhibitor and each of the remaining therapies: ARB (OR 1.10; 95% CrI 0.90–1.40), ACE inhibitor plus ARB (0.97; 95% CrI 0.72–1.29), DR inhibitor plus ACE inhibitor (0.99; 95% CrI 0.65–1.57), and DR inhibitor plus ARB (1.18; 95% CrI 0.78–1.84). No significant differences were showed between ACE inhibitors and ARBs with respect to all-cause mortality, cardiovascular mortality, myocardial infarction, stroke, angina pectoris, hospitalization for heart failure, ESRD, or doubling serum creatinine. Findings were limited by the clinical and methodological heterogeneity of the included studies. Potential inconsistency was identified in network meta-analyses of stroke and angina pectoris, limiting the conclusiveness of findings for these single endpoints.

Conclusions

In adults with diabetes, comparisons of different RAS blockers showed similar effects of ACE inhibitors and ARBs on major cardiovascular and renal outcomes. Compared with monotherapies, the combination of an ACE inhibitor and an ARB failed to provide significant benefits on major outcomes. Clinicians should discuss the balance between benefits, costs, and potential harms with individual diabetes patients before starting treatment.

Review registration

PROSPERO CRD42014014404     

Quaternary assembly and crystal structure of GDP-D-mannose 4,6 dehydratase from Paramecium bursaria Chlorella virus

GDP-D-mannose 4,6 dehydratase is the first enzyme in the de novo biosynthetic pathway of GDP-L-fucose, the activated form of L-fucose, a monosaccharide found in organisms ranging from bacteria to mammals. We determined the three-dimensional structure of GDP-D-mannose 4,6 dehydratase from the Paramecium bursaria Chlorella virus at 3.8A resolution. Unlike other viruses that use the host protein machinery to glycosylate their proteins, P. bursaria Chlorella virus modifies its structural proteins using many glycosyltransferases, being the first virus known to encode enzymes involved in sugar metabolism. P. bursaria Chlorella virus GDP-D-mannose 4,6 dehydratase belongs to the short-chain dehydrogenase/reductase protein superfamily. Accordingly, the family fold and the specific Thr, Tyr, and Lys catalytic triad are well conserved in the viral enzyme.     

Structure, conformational stability, and enzymatic properties of acylphosphatase from the hyperthermophile Sulfolobus solfataricus

The structure of AcP from the hyperthermophilic archaeon Sulfolobus solfataricus has been determined by (1)H-NMR spectroscopy and X-ray crystallography. Solution and crystal structures (1.27 A resolution, R-factor 13.7%) were obtained on the full-length protein and on an N-truncated form lacking the first 12 residues, respectively. The overall Sso AcP fold, starting at residue 13, displays the same betaalphabetabetaalphabeta topology previously described for other members of the AcP family from mesophilic sources. The unstructured N-terminal tail may be crucial for the unusual aggregation mechanism of Sso AcP previously reported. Sso AcP catalytic activity is reduced at room temperature but rises at its working temperature to values comparable to those displayed by its mesophilic counterparts at 25-37 degrees C. Such a reduced activity can result from protein rigidity and from the active site stiffening due the presence of a salt bridge between the C-terminal carboxylate and the active site arginine. Sso AcP is characterized by a melting temperature, Tm, of 100.8 degrees C and an unfolding free energy, DeltaG(U-F)H2O, at 28 degrees C and 81 degrees C of 48.7 and 20.6 kJ mol(-1), respectively. The kinetic and structural data indicate that mesophilic and hyperthermophilic AcP's display similar enzymatic activities and conformational stabilities at their working conditions. Structural analysis of the factor responsible for Sso AcP thermostability with respect to mesophilic AcP's revealed the importance of a ion pair network stabilizing particularly the beta-sheet and the loop connecting the fourth and fifth strands, together with increased density packing, loop shortening and a higher alpha-helical propensity.