Haemoglobin mass and running time trial performance after recombinant human erythropoietin administration in trained men

<p>Recombinant human erythropoietin (rHuEpo) increases haemoglobin mass (Hbmass) and maximal oxygen uptake (v˙ O2 max).</p> <p>Purpose: This study defined the time course of changes in Hbmass, v˙ O2 max as well as running time trial performance following 4 weeks of rHuEpo administration to determine whether the laboratory observations would translate into actual improvements in running performance in the field.</p> <p>Methods: 19 trained men received rHuEpo injections of 50 IUNkg21 body mass every two days for 4 weeks. Hbmass was determined weekly using the optimized carbon monoxide rebreathing method until 4 weeks after administration. v˙ O2 max and 3,000 m time trial performance were measured pre, post administration and at the end of the study.</p> <p>Results: Relative to baseline, running performance significantly improved by ,6% after administration (10:3061:07 min:sec vs. 11:0861:15 min:sec, p,0.001) and remained significantly enhanced by ,3% 4 weeks after administration (10:4661:13 min:sec, p,0.001), while v˙ O2 max was also significantly increased post administration (60.765.8 mLNmin21Nkg21 vs. 56.066.2 mLNmin21Nkg21, p,0.001) and remained significantly increased 4 weeks after rHuEpo (58.065.6 mLNmin21Nkg21, p = 0.021). Hbmass was significantly increased at the end of administration compared to baseline (15.261.5 gNkg21 vs. 12.761.2 gNkg21, p,0.001). The rate of decrease in Hbmass toward baseline values post rHuEpo was similar to that of the increase during administration (20.53 gNkg21Nwk21, 95% confidence interval (CI) (20.68, 20.38) vs. 0.54 gNkg21Nwk21, CI (0.46, 0.63)) but Hbmass was still significantly elevated 4 weeks after administration compared to baseline (13.761.1 gNkg21, p<0.001).</p> <p>Conclusion: Running performance was improved following 4 weeks of rHuEpo and remained elevated 4 weeks after administration compared to baseline. These field performance effects coincided with rHuEpo-induced elevated v˙ O2 max and Hbmass.</p&gt

Cation binding and conformational changes in VILIP and NCS-1, two neuron-specific calcium-binding proteins

VILIP and NCS-1, neural-specific, 22-kDa Ca(2+)-binding proteins possessing four EF-hands, were expressed in Escherichia coli to study their divalent cation properties. Flow dialysis (Ca2+ binding) and equilibrium gel filtration (Mg2+ binding) revealed that both recombinant proteins possess only two active metal-binding sites, which can accommodate either Ca2+ or Mg2+. VILIP binds cations without cooperativity with intrinsic affinity constants K'Ca of 1.0 x 10(6) M-1 and K'Mg of 4.8 x 10(3) M-1.Mg2+ antagonizes Ca2+ binding by shifting the isotherms to higher free Ca2+ concentrations without changing their shape. The competition equation yields a K'Mg, comp value of 180 M-1 for both sites. NCS-1 binds two Mg2+ without cooperativity with K'Mg of 8.3 x 10(4) M-1 and two Ca2+ with very strong positive cooperativity (nH = 1.96). In the absence of Mg2+ the K'Ca1 and K'Ca2 values are 8.9 x 10(4) and 1.4 x 10(8) M-1, respectively, which represent an allosteric increase of 1600-fold. Mg2+ shifts the Ca(2+)-binding isotherms to higher Ca2+ concentrations, yielding a K'Mg, comp value of 800 M-1 for both sites. Thus VILIP and NCS-1 show three remarkable differences in the Ca2+/Mg2+ binding parameters: 1) VILIP binds Ca2+ with much lower affinity than NCS-1; 2) VILIP binds Ca2+ in a noncooperative way, whereas NCS-1 shows maximal positive cooperativity; 3) in VILIP the Mg2+/Ca2+ antagonism is much weaker than in NCS-1. Conformational changes monitored by Trp fluorescence indicate that the metal-free forms already are highly structured. Ca2+ binding promotes a 20-30% increase of fluorescence in both proteins, but whereas the Mg2+ form of VILIP has the same fluorescence properties as the metal-free form, Mg(2+)-saturated NCS-1 has those of the Ca2+ form. Near UV difference spectra confirmed that in VILIP the Mg2+ form is very similar to the metal-free form; in NCS-1 it is different, especially in the Tyr region. NCS-1 possesses one unique Cys-38 in EF-hand site I. Its reactivity (kSH) toward 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) is the same for the Ca(2+)- and Mg(2+)-loaded protein, but kSH is 4-fold higher in metal-free NCS-1. VILIP possesses two additional thiols, one of which is inaccessible to DTNB in the native protein. The reactivity of the two accessible thiols is identical in the metal-free and Mg2+ forms and 5-fold higher than in the Ca2+ form

The added value of a micro-level ecological approach when mapping self-regulatory control processes and externalizing symptoms during adolescence: a systematic review.

Deficits in self-regulatory control (SRC) represent a core characteristic of externalizing (EXT) symptoms (e.g., rule-breaking behavior or aggressive behaviors) in adolescents. This review aims to specify the added value of ecologically valid assessments at a micro-level when examining the associations between SRC and EXT symptoms in adolescents. This systematic review was reported according to the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) 2020. The search strategy addressed the added value of (1) naturalistic assessment for the understanding of the relationship between (2) SRC and (3) EXT symptoms in (4) adolescents. We conducted comprehensive searches in bibliographic databases. An additional search was conducted in Google Scholar and supplementary studies were identified through backward and forward citation tracking. Twenty-four studies (n = 4071 adolescents) met the inclusion criteria. The methods used to assess naturalistic aspects included the experience sampling method (ecological momentary- or ambulatory assessment) and the time-course approach (i.e., real-time assessment of SRC processes referring to situations approximating real-life experience where SRC are to be engaged such as in frustrating situations). Micro-level ecological assessments, when mapping the intra-individual relationships between SRC processes and EXT symptoms over time in adolescents within their natural context (i.e., real world) of expression in real time, added a finer-grained observation alongside with a higher ecological validity. Micro-level approaches may enhance the understanding of the complex interplay between SRC and EXT symptoms in adolescence, especially in interventional studies, allowing for the acquisition of endpoints with a higher relevance for everyday functioning

Remodeling of the AB site of rat parvalbumin and oncomodulin into a canonical EF-hand

Parvalbumin (PV) and the homologous protein oncomodulin (OM) contain three EF-hand motifs, but the first site (AB) cannot bind Ca2+. Here we aimed to recreate the putative ancestral proteins [D19-28E]PV and [D19-28E]OM by replacing the 10-residue-long nonfunctional loop in the AB site by a 12-residue canonical loop. To create an optical conformational probe we also expressed the homologs with a F102W replacement. Unexpectedly, in none of the proteins did the mutation reactivate the AB site. The AB-remodeled parvalbumins bind two Ca2+ ions with strong positive cooperativity (nH = 2) and moderate affinity ([Ca2+]0.5 = 2 microM), compared with [Ca2+]0.5 = 37 nM and nH = 1 for the wild-type protein. Increasing Mg2+ concentrations changed nH from 2 to 0.65, but without modification of the [Ca2+]0. 5-value. CD revealed that the Ca2+ and Mg2+ forms of the remodeled parvalbumins lost one-third of their alpha helix content compared with the Ca2+ form of wild-type parvalbumin. However, the microenvironment of single Trp residues in the hydrophobic cores, monitored using intrinsic fluorescence and difference optical density, is the same. The metal-free remodeled parvalbumins possess unfolded conformations. The AB-remodeled oncomodulins also bind two Ca2+ with [Ca2+]0.5 = 43 microM and nH = 1.45. Mg2+ does not affect Ca2+ binding. Again the Ca2+ forms display two-thirds of the alpha-helical content in the wild-type, while their core is still strongly hydrophobic as monitored by Trp and Tyr fluorescence. The metal-free oncomodulins are partially unfolded and seem not to possess a hydrophobic core. Our data indicate that AB-remodeled parvalbumin has the potential to regulate cell functions, whereas it is unlikely that [D19-28E]OM can play a regulatory role in vivo. The predicted evolution of the AB site from a canonical to an abortive EF-hand may have been dictated by the need for stronger interaction with Mg2+ and Ca2+, and a high conformational stability of the metal-free forms