Adenylate effects on protein phosphorylation in the interenvelope lumen of pea chloroplasts

A 64-kilodalton (kDa) protein, situated in the lumen between the inner and outer envelopes of pea (Pisum sativum L.) chloroplasts (Soll and Bennett 1988, Eur. J. Biochem., 175, 301–307) is shown to undergo reversible phosphorylation in isolated mixed envelope vesicles. It is the most conspicuously labelled protein after incubation of envelopes with 33 nmol·1-1 [-32P]ATP whereas incubation with 50 mol·1-1 [-32P]ATP labels most prominently two outer envelope proteins (86 and 23 kDa). Half-maximum velocity for phosphorylation of the 64-kDa protein occurs with 200 nmol·1-1 ATP, and around 40 mol·1-1 ATP for phosphorylation of the 86- and 23-kDa proteins, indicating the operation of two distinct kinases. GGuanosine-, uridine-, cytidine 5-triphosphate and AMP are poor inhibitors of the labelling of the 64-kDa protein with [-32P]ATP. On the other hand, ADP has a potent influence on the extent of labelling (half-maximal inhibition at 1–5 mol·1-1). The ADP-dependent appearance of 32P in ATP indicates that ADP acts by reversal of kinase activity and not as a competitive inhibitor. However, the most rapid loss of 32P from pre-labelled 64-kDa protein occurs when envelope vesicles are incubated with ATP t1/2=15 s at 20 molsd1-1 ATP). This induced turnover of phosphate appears to be responsible for the rapid phosphoryl turnover seen in situ

Neuromuscular synaptic function in mice lacking major subsets of gangliosides

Gangliosides are a family of sialylated glycosphingolipids enriched in the outer leaflet of neuronal membranes, in particular at synapses. Therefore, they have been hypothesized to play a functional role in synaptic transmission. We have measured in detail the electrophysiological parameters of synaptic transmission at the neuromuscular junction (NMJ) ex vivo of a GD3-synthase knockout mouse, expressing only the O- and a-series gangliosides, as well as of a GM2/GD2-synthase*GD3-synthase double-knockout (dKO) mouse, lacking all gangliosides except GM3. No major synaptic deficits were found in either null-mutant. However, some extra degree of rundown of acetylcholine release at high intensity use was present at the dKO NMJ and a temperature-specific increase in acetylcholine release at 35 °C was observed in GD3-synthase knockout NMJs, compared with wild-type. These results indicate that synaptic transmission at the NMJ is not crucially dependent on the particular presence of most ganglioside family members and remains largely intact in the sole presence of GM3 ganglioside. Rather, presynaptic gangliosides appear to play a modulating role in temperature- and use-dependent fine-tuning of transmitter output

Structural plasticity and in vivo activity of Cas1 from the type I-F CRISPR-Cas system

CRISPR-Cas systems are adaptive immune systems in prokaryotes that provide protection against viruses and other foreign DNA. In the adaptation stage, foreign DNA is integrated into CRISPR (clustered regularly interspaced short palindromic repeat) arrays as new spacers. These spacers are used in the interference stage to guide effector CRISPR associated (Cas) protein(s) to target complementary foreign invading DNA. Cas1 is the integrase enzyme that is central to the catalysis of spacer integration. There are many diverse types of CRISPR-Cas systems, including type I-F systems, which are typified by a unique Cas1-Cas2-3 adaptation complex. In the present study we characterize the Cas1 protein of the potato phytopathogen Pectobacterium atrosepticum, an important model organism for understanding spacer acquisition in type I-F CRISPR-Cas systems. We demonstrate by mutagenesis that Cas1 is essential for adaptation in vivo and requires a conserved aspartic acid residue. By X-ray crystallography, we show that although P. atrosepticum Cas1 adopts a fold conserved among other Cas1 proteins, it possesses remarkable asymmetry as a result of structural plasticity. In particular, we resolve for the first time a flexible, asymmetric loop that may be unique to type I-F Cas1 proteins, and we discuss the implications of these structural features for DNA binding and enzymatic activity

Tuning Fermi level within half-metallic gap in Co-based Heusler alloys

We systematically study the tuning of Fermi level in half-metal gap for Co-based full-Heusler alloys in the frame of first-principles calculations. It is found that the position of the Fermi level within whole gap can be specified in compounds Co2MnZ11-x Z2x (Z1 and Z2 are the III, IV or V main group element). As one of examples, Co2MnAl doped by As is further explored in detail, of which the Fermi level falls into the expectative position of the minority spin gap at proper concentration of As doping (0.5 < x < 0.75). This can effectively suppress the so-called spin-flip excitation and promise a good candidate for spintronics application

The prognostic value of estimated glomerular filtration rate, amino-terminal portion of the pro-hormone B-type natriuretic peptide and parameters of cardiopulmonary exercise testing in patients with chronic heart failure

The aim of this study was to evaluate the prognostic value of renal function in relation to amino-terminal portion of the pro-hormone B-type natriuretic peptide (NT-proBNP) and parameters of cardiopulmonary exercise testing in predicting mortality and morbidity in patients with moderate chronic heart failure (CHF). Sixty-one CHF patients were included in the study. Patients’ characteristics were: age 64.3±11.6 years; New York Heart Association class I/II/III: 14/37/10; left ventricular ejection fraction: 0.30±0.13 (%); NT-proBNP: 252.2±348.0 (ng/L); estimated creatinine clearance (e-CC): 73.6±31.4 (mL/min); estimated glomerular filtration rate (e-GFR): 66.1±24.6 (mL/min/1.73 m2); the highest O2 uptake during exercise (VO2-peak): 1.24±0.12 mL/kg/min; VO2/workload: 8.52±1.81 (mL/min/W)]. During follow up (59.5±4.0 months) there were 15 cardiac deaths and 16 patients were hospitalized due to progression of heart failure. NT-proBNP and VO2/workload were independently associated with cardiac death (P=0.007 and P=0.006, respectively). Hospitalization for progressive CHF was only associated with NT-proBNP (P=0.002). The combined cardiac events (cardiac death and hospitalization) were associated with NT-proBNP and VO2/workload (P=0.007 and P=0.005, respectively). The addition of estimates of renal function (neither serum creatinine nor e-GFR) did not improve the prognostic value for any of the models. In conclusion, in patients with moderate CHF, increased NT-proBNP and reduced VO2/workload identify those with increased mortality and morbidity, irrespective of estimates of renal function