Confocal endomicroscopy of neuromuscular junctions stained with physiologically inert protein fragments of tetanus toxin

Live imaging of neuromuscular junctions (NMJs) in situ has been constrained by the suitability of ligands for inert vital staining of motor nerve terminals. Here, we constructed several truncated derivatives of the tetanus toxin C-fragment (TetC) fused with Emerald Fluorescent Protein (emGFP). Four constructs, namely full length emGFP-TetC (emGFP-865:TetC) or truncations comprising amino acids 1066–1315 (emGFP-1066:TetC), 1093–1315 (emGFP-1093:TetC) and 1109–1315 (emGFP-1109:TetC), produced selective, high-contrast staining of motor nerve terminals in rodent or human muscle explants. Isometric tension and intracellular recordings of endplate potentials from mouse muscles indicated that neither full-length nor truncated emGFP-TetC constructs significantly impaired NMJ function or transmission. Motor nerve terminals stained with emGFP-TetC constructs were readily visualised in situ or in isolated preparations using fibre-optic confocal endomicroscopy (CEM). emGFP-TetC derivatives and CEM also visualised regenerated NMJs. Dual-waveband CEM imaging of preparations co-stained with fluorescent emGFP-TetC constructs and Alexa647-α-bungarotoxin resolved innervated from denervated NMJs in axotomized WldS mouse muscle and degenerating NMJs in transgenic SOD1G93A mouse muscle. Our findings highlight the region of the TetC fragment required for selective binding and visualisation of motor nerve terminals and show that fluorescent derivatives of TetC are suitable for in situ morphological and physiological characterisation of healthy, injured and diseased NMJs

Functions of the shared N-terminus of slow wallerian degeneration (WldS) and Ube4b proteins: a sequence required for axon protection and ubiquitination

5th FORUM OF EUROPEAN NEUROSCIENCE; Vienna (Austria) - abstract autho

Solution structure of the active-centre mutant I14A of the histidine-containing phosphocarrier protein from Staphylococcus carnosus

High-pressure NMR experiments performed on the histidine-containing phosphocarrier protein (HPr) from Staphylococcus carnosus have shown that residue Ile14, which is located in the active-centre loop, exhibits a peculiarly small pressure response. In contrast, the rest of the loop shows strong pressure effects as is expected for typical protein interaction sites. To elucidate the structural role of this residue, the mutant protein HPr(I14A), in which Ile14 is replaced by Ala, was produced and studied by solution NMR spectroscopy. On the basis of 1406 structural restraints including 20 directly detected hydrogen bonds, 49 1H(N)-15N, and 25 1H(N)-1Halpha residual dipolar couplings, a well resolved three-dimensional structure could be determined. The overall fold of the protein is not influenced by the mutation but characteristic conformational changes are introduced into the active-centre loop. They lead to a displacement of the ring system of His15 and a distortion of the N-terminus of the first helix, which supports the histidine ring. In addition, the C-terminal helix is bent because the side chain of Leu86 located at the end of this helix partly fills the hydrophobic cavity created by the mutation. Xenon, which is known to occupy hydrophobic cavities, causes a partial reversal of the mutation-induced structural effects. The observed structural changes explain the reduced phosphocarrier activity of the mutant and agree well with the earlier suggestion that Ile14 represents an anchoring point stabilizing the active-centre loop in its correct conformation

Indução do estro no pós-parto em vacas primíparas Holandês-Zebu Induction of estrus in the postpartum of Holstein-Zebu heifers through norgestomet

Avaliou-se o efeito do peso corporal no início do tratamento com progestágeno sobre as características reprodutivas de vacas mestiças Holandês-Zebu no pós-parto. Foram utilizadas 64 vacas, divididas em quatro grupos: GI - vacas com peso corporal entre 390-458kg e submetidas a tratamento hormonal com norgestomet, GII - vacas com peso corporal entre 464-562kg e submetidas a tratamento hormonal com norgestomet, GIII - vacas com peso corporal entre 374-451kg (controle) e GIV - vacas com peso corporal entre 452-545kg (controle). Os animais do grupo II manifestaram o primeiro estro no pós-parto mais cedo que os demais (64,4 dias - GII vs. 109,4-GI; 143,2-GIII e 105,1-GIV dias), e apresentaram menor período de serviço (94,6 dias vs. 125,5; 160,9 e 131,0 dias, na mesma ordem de citação anterior). Quanto às taxas de manifestação de estro e de gestação final, não se verificaram diferenças (P>0,05) entre os tratamentos. Os animais do GII apresentaram o menor período de serviço e os do GIII, o maior (94,6 vs. 160,9). Não houve influência do tratamento hormonal nem do peso corporal sobre a produção de leite e duração da lactação. O uso do implante de progestágeno nos animais que apresentaram maiores peso e condição corporal no início do tratamento respondeu por menor intervalo entre o parto e o primeiro estro. O uso do progestágeno em animais mais leves esteve associado ao retorno mais rápido à atividade ovariana cíclica no pós-parto.<br>The experiment was carried out to evaluate the effect of two ranges of body weight and norgestomet treatment on the reproductive parameters of postpartum crossbred Holstein-zebu cows. Sixty four primiparous cows were randomly allocated to four treatments 40 days after calving: group I - cows with body weight ranging from 390 to 458kg and norgestomet treated; group II - cows with body weight ranging from 464 to 562kg and norgestomet treated; group III - cows with body weight ranging from 390 to 458kg (control); and group IV - cows with body weight ranging from 464 to 562kg (control). Progestagen auricular implants were mantained during 10 days and the cows were mated to bulls submitted to breeding soundness evaluation. Animals from treatment II showed estrus earlier than animals of the others treatments (II: 64.4; I: 109.4; III: 143.2 and IV: 105.1 days; P<0.05), and shorter open days (II: 94.6; I: 125.5; III: 160.9 and IV: 131.0 days; P<0.05). Estrus and pregnance rates did not differ between treatments (P>0.05). The hormonal treatment and the body weight did not affect the total and daily milk yield, and length of lactation (P>0.05). Progestagen treated, heavier and better body condition scored animals had shorter open days, and returned to postpartum ovarian ciclicity faster than lighter animals