Distinct Properties of Hexameric but Functionally Conserved Mycobacterium tuberculosis Transcription-Repair Coupling Factor

Transcription coupled nucleotide excision repair (TC-NER) is involved in correcting UV-induced damage and other road-blocks encountered in the transcribed strand. Mutation frequency decline (Mfd) is a transcription repair coupling factor, involved in repair of template strand during transcription. Mfd from M. tuberculosis (MtbMfd) is 1234 amino-acids long harboring characteristic modules for different activities. Mtbmfd complemented Escherichia coli mfd (Ecomfd) deficient strain, enhanced survival of UV irradiated cells and increased the road-block repression in vivo. The protein exhibited ATPase activity, which was stimulated ∼1.5-fold in the presence of DNA. While the C-terminal domain (CTD) comprising amino acids 630 to 1234 showed ∼2-fold elevated ATPase activity than MtbMfd, the N-terminal domain (NTD) containing the first 433 amino acid residues was able to bind ATP but deficient in hydrolysis. Overexpression of NTD of MtbMfd led to growth defect and hypersensitivity to UV light. Deletion of 184 amino acids from the C-terminal end of MtbMfd (MfdΔC) increased the ATPase activity by ∼10-fold and correspondingly exhibited efficient translocation along DNA as compared to the MtbMfd and CTD. Surprisingly, MtbMfd was found to be distributed in monomer and hexamer forms both in vivo and in vitro and the monomer showed increased susceptibility to proteases compared to the hexamer. MfdΔC, on the other hand, was predominantly monomeric in solution implicating the extreme C-terminal region in oligomerization of the protein. Thus, although the MtbMfd resembles EcoMfd in many of its reaction characteristics, some of its hitherto unknown distinct properties hint at its species specific role in mycobacteria during transcription-coupled repair

Contribution of Social Isolation, Restraint, and Hindlimb Unloading to Changes in Hemodynamic Parameters and Motion Activity in Rats

The most accepted animal model for simulation of the physiological and morphological consequences of microgravity on the cardiovascular system is one of head-down hindlimb unloading. Experimental conditions surrounding this model include not only head-down tilting of rats, but also social and restraint stresses that have their own influences on cardiovascular system function. Here, we studied levels of spontaneous locomotor activity, blood pressure, and heart rate during 14 days under the following experimental conditions: cage control, social isolation in standard rat housing, social isolation in special cages for hindlimb unloading, horizontal attachment (restraint), and head-down hindlimb unloading. General activity and hemodynamic parameters were continuously monitored in conscious rats by telemetry. Heart rate and blood pressure were both evaluated during treadmill running to reveal cardiovascular deconditioning development as a result of unloading. The main findings of our work are that: social isolation and restraint induced persistent physical inactivity, while unloading in rats resulted in initial inactivity followed by normalization and increased locomotion after one week. Moreover, 14 days of hindlimb unloading showed significant elevation of blood pressure and slight elevation of heart rate. Hemodynamic changes in isolated and restrained rats largely reproduced the trends observed during unloading. Finally, we detected no augmentation of tachycardia during moderate exercise in rats after 14 days of unloading. Thus, we concluded that both social isolation and restraint, as an integral part of the model conditions, contribute essentially to cardiovascular reactions during head-down hindlimb unloading, compared to the little changes in the hydrostatic gradient

Short-term but not long-term adrenalectomy modulates amplitude and frequency of the CRH41 episodic release in push-pull cannulated median eminence of free-moving rats.

International audienceCRH 41 release in push-pull cannulated median eminence (ME) was measured in unanesthetized male rats, 3 and 7 days after adrenalectomy (ADX) and in sham-lesioned controls. Perfusion started at 13.30 h and perfusate samples were collected at 5 min intervals for 3 h to estimate the mean release rate of CRH41. The major parameters of the neurohormone's episodic release pattern were analyzed using the Ultra algorithm. In a parallel study, 3 groups of similarly treated rats were used to measure plasma ACTH and hypothalamic CRH41. Three days after ADX, the plasma ACTH titers had risen 14-fold, the hypothalamic CRH41 content had decreased by 40%, while the CRH41 release in the ME had doubled as a result of a significant increase in most variables of the pulsatile release pattern: pulse frequency (+34%; P < 0.01), mean amplitude (+36%; P < 0.05), mean peak levels (+67%; P < 0.01) and mean pulse nadirs (x2.5; P < 0.01). Seven days after ADX, even though plasma ACTH had further increased to 30-times control levels, hypothalamic CRH41 content and CRH41 release in the ME had returned to almost control levels. The possible mechanisms of the discrepancy between the CRH and ACTH response time-courses following ADX are discussed

Evidence for an age-dependent decrease in the immunoreactive prolactin-containing terminals of the median eminence of male rats.

International audienceLabelling patterns of immunoreactive prolactin (IR-PRL)-containing and tyrosine hydroxylase (TH)-containing nerve terminals of the median eminence (ME) were compared in young adult (aged 3 months) and old (aged 24 months) male Wistar rats. In the young rats, IR-PRL- and TH-immunostained fibres extended throughout the external most layer of the ME. In the old rats, a significant decrease in the intensity of labelling of IR-PRL terminals was observed in this layer, with a slight reduction in the extent of labelling. As far as TH terminals were concerned, no difference could be detected between young and old animals

A subpopulation of corticotropin-releasing hormone neurosecretory cells in the paraventricular nucleus of the hypothalamus also contain NADPH-diaphorase.

International audienceThe coexistence of ND with CRH 41 was explored in the parvicellular neurons of the PVN, using dual histochemical and radioimmunocytochemical labelling with the light microscope, in rats treated with colchicine. Even though the ND staining was scarce, a clear colocalization was evidenced in the parvicellular part of the PVN. Under these conditions, the ratio of neurons expressing both markers, ND and CRH, amounted about 15% of the CRH-containing neuron population. This result provides a useful tool to study morphological plastic changes in the PVN in response to environmental variations