Effect of length and caffeine on isometric tetanus relaxation of frog sartorius muscles

In an isometric tetanus of frog sartorius muscle the total relaxation time increased linearly with change in length from 0.7 to 1.4 times rest length. Maximal rate of relaxation, measured from the time derivative (dp/dt) of tension decay, decreased with both decrease and increase from rest length in correlation with the generated tetanus tension. Stretching the muscle did not significantly affect the times to maximal rate, positive and negative inflexion points but greatly increased the time to total relaxation from the negative inflexion point. Caffeine at 2 mM, acting on muscles at rest length, also slowed the relaxation and decreased the maximal rate of tension decay. However, caffeine increased the times to maximal rate, positive and negative inflexion points without significantly affecting time to total relaxation from the negative inflexion point. These results suggest that caffeine slows an earlier step in relaxation, while stretch slows a later step. It is proposed that muscle relaxation is a two step process: an initial step that is regulated by the rate of Ca2+ uptake by sarcoplasmic reticulum, and a later step that is mostly controlled by the speed of dissociation of remaining cross-bridges.     

Reconstructing Indian-Australian phylogenetic link

Background  

An early dispersal of biologically and behaviorally modern humans from their African origins to Australia, by at least 45 thousand years via southern Asia has been suggested by studies based on morphology, archaeology and genetics. However, mtDNA lineages sampled so far from south Asia, eastern Asia and Australasia show non-overlapping distributions of haplogroups within pan Eurasian M and N macrohaplogroups. Likewise, support from the archaeology is still ambiguous.     

Sequential Elution Interactome Analysis of the Mind Bomb 1 Ubiquitin Ligase Reveals a Novel Role in Dendritic Spine Outgrowth

The mind bomb 1 (Mib1) ubiquitin ligase is essential for controlling metazoan development by Notch signaling and possibly the Wnt pathway. It is also expressed in postmitotic neurons and regulates neuronal morphogenesis and synaptic activity by mechanisms that are largely unknown. We sought to comprehensively characterize the Mib1 interactome and study its potential function in neuron development utilizing a novel sequential elution strategy for affinity purification, in which Mib1 binding proteins were eluted under different stringency and then quantified by the isobaric labeling method. The strategy identified the Mib1 interactome with both deep coverage and the ability to distinguish high-affinity partners from low-affinity partners. A total of 817 proteins were identified during the Mib1 affinity purification, including 56 high-affinity partners and 335 low-affinity partners, whereas the remaining 426 proteins are likely copurified contaminants or extremely weak binding proteins. The analysis detected all previously known Mib1-interacting proteins and revealed a large number of novel components involved in Notch and Wnt pathways, endocytosis and vesicle transport, the ubiquitin-proteasome system, cellular morphogenesis, and synaptic activities. Immunofluorescence studies further showed colocalization of Mib1 with five selected proteins: the Usp9x (FAM) deubiquitinating enzyme, alpha-, beta-, and delta-catenins, and CDKL5. Mutations of CDKL5 are associated with early infantile epileptic encephalopathy-2 (EIEE2), a severe form of mental retardation. We found that the expression of Mib1 down-regulated the protein level of CDKL5 by ubiquitination, and antagonized CDKL5 function during the formation of dendritic spines. Thus, the sequential elution strategy enables biochemical characterization of protein interactomes; and Mib1 analysis provides a comprehensive interactome for investigating its role in signaling networks and neuronal development.Mind bomb 1 (Mib1)¹, an E3 ubiquitin ligase, is a critical regulator of metazoan development with a large, and ever expanding, number of functions through interactions with a variety of protein partners. Mib1 mutants were first found in zebrafish mutagenesis screens (1), in which the mutants had neurogenic defects, most notably supernumerary primary neurons, and additional deficits in the development of somites (2), ear (3), and vasculature (4). These phenotypes are predominantly the consequences of impaired Notch signaling, as Mib1 is an essential activator of Notch Delta/Serrate/lag-2 (DSL) ligands (1). Mib1 also controls the development of several other organ and tissue systems, including gastrointestinal tract (5), limb bud (6), and the immune system (7). Mib1 is highly conserved across species. For instance, zebrafish Mib1 protein is 68%, 94%, and 94% identical to its fly, mouse, and human orthologs, respectively (8). Moreover, Mib1 has a paralog (Mib2) that shares 38% identical protein sequence with Mib1 in mouse (9). Mib2 is only abundantly expressed in adult tissue, however, and thus does not function in early development. Consistently, Mib1 knockout in mice results in embryonic mortality (10), whereas Mib2 deletion has no obvious effect on mouse development (6).In addition to its role in cell fate determination during early development, Mib1 is also abundantly expressed in the adult brain (11) and plays an important role in neuronal morphogenesis (12). Neurons usually have two basic polarized structures, a single extended axon for sending signals and multiple branched dendrites (or more precisely, the somatodendritic compartment) for receiving signals. Many principle neurons in mammals further grow dendritic spines that are tiny protrusions extended from dendritic branches, creating local postsynaptic compartments for the formation of excitatory synapses. In these synapses, the postsynaptic density (PSD) is an electron-dense membrane thickening aligned with the presynaptic active zone at synaptic junctions. During neuronal morphogenesis, axonal growth and path finding (13), dendrite formation (14), dendritic spine assembly (15), and synaptogenesis (16) are independent but highly related processes controlled by genetic elements and environmental cues. Although dramatic progress has been made in identifying the signaling cascades responsible for these processes, large gaps still remain in the connection of individual signaling components as well as in the coordination of multiple pathways. Our previous proteomics analysis identified that Mib1 is highly enriched in the PSD fraction, and regulates neurite outgrowth in postmitotic neurons (12). Mib1 conditional knockout mouse studies suggest a role in long-term potentiation (LTP) and synaptic plasticity (11), and further intriguing actions of Mib1 continue to be discovered. Mib1 was found to mediate the degradation of survival motor neuron 1 (SMN1), which contributes to spinal muscular atrophy (17). Mib1 was reported to be essential for Wnt3A activation of beta-catenin signaling through the receptor RYK (18), and a recent yeast two-hybrid screen indicated that Mib1 interacts with 81 candidate proteins beyond the canonical Notch pathway (19). The ongoing identification of new Mib1 interaction partners and functions underscores the need to characterize the Mib1 interactome en masse with high confidence.The combination of affinity purification and liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a powerful method for analyzing protein interaction networks. Technological advances in LC-MS/MS have continually increased the sensitivity of protein detection (20, 21), allowing for the analysis of complex samples (22). The primary advantage of this technique, however, has also proven to be its greatest weakness: without stringent washes and data filtering, a vast number of false positives are included in the resulting data sets (23). Methods such as tandem-affinity purification (24) have been developed to remove nonspecific contaminants, but two-step purification requires large quantities of starting materials and reduces sensitivity to loosely bound proteins. Removing contaminants by buffers containing high concentrations of salt and detergents can help limit false positives, but a delicate balance lies between rinsing contaminants and losing weakly bound but true interaction partners, and thus inflating false negative results. In addition, in vivo crosslinking and quantitative analysis are used to enhance the capture of transient interacting proteins (25, 26).To this end, we attempted to characterize the Mib1 interactome by combining glutathione S-transferase (GST) protein affinity purification and advanced quantitative mass spectrometry. In our sequential elution strategy, Mib1 interaction partners were bound to affinity resins coated with GST-Mib1 domains, then eluted in three sequential buffers of increasing stringency. Proteins in these three eluents were identified and quantified by an isobaric labeling Tandem Mass Tag (TMT) method (15). The elution profile of each protein reflected its binding affinity to the GST-Mib1 resins. The strategy not only provides high sensitivity to recover weakly bound partners, but also allows for the affinity-based classification of the interactome and the removal of contaminants. By this approach, we were able to recover 817 putative Mib1 binding partners in adult rat brain and accepted about half of the proteins with high confidence. This study also uncovered that Mib1 interacts with CDKL5, a protein kinase implicated in early infantile epileptic encephalopathy-2 (EIEE2), a severe form of epilepsy and mental retardation in females (28). We then found that Mib1 acts to down-regulate CDKL5 and inhibits its promotion of dendritic spine outgrowth.     

Exercise Does Not Protect against MPTP-Induced Neurotoxicity in BDNF Happloinsufficent Mice

Exercise has been demonstrated to potently protect substantia nigra pars compacta (SN) dopaminergic neurons from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity. One mechanism proposed to account for this neuroprotection is the upregulation of neurotrophic factors. Several neurotrophic factors, including Brain Derived Neurotrophic Factor (BDNF), have been shown to upregulate in response to exercise. In order to determine if exercise-induced neuroprotection is dependent upon BDNF, we compared the neuroprotective effects of voluntary exercise in mice heterozygous for the BDNF gene (BDNF+/-) with strain-matched wild-type (WT) mice. Stereological estimates of SNpc DA neurons from WT mice allowed 90 days exercise via unrestricted running demonstrated complete protection against the MPTP-induced neurotoxicity. However, BDNF+/- mice allowed 90 days of unrestricted exercise were not protected from MPTP-induced SNpc DA neuron loss. Proteomic analysis comparing SN and striatum from 90 day exercised WT and BDNF+/- mice showed differential expression of proteins related to energy regulation, intracellular signaling and trafficking. These results suggest that a full genetic complement of BDNF is critical for the exercise-induced neuroprotection of SNpc DA neurons.     

Imaging caffeine-induced Ca2+ transients in individual fast-twitch and slow-twitch rat skeletal muscle fibers

Fast-twitch and slow-twitch rat skeletal muscles producedissimilar contractures with caffeine. We used digital imagingmicroscopy to monitor Ca²⁺ (withfluo 3-acetoxymethyl ester) and sarcomere motion in intact, unrestrained rat muscle fibers to study this difference. Changes inCa²⁺ in individual fibers weremarkedly different from average responses of a population. All fibersshowed discrete, nonpropagated, local Ca²⁺ transients occurring randomlyin spots about one sarcomere apart. Caffeine increased localCa²⁺ transients and sarcomeremotion initially at 4 mM in soleus and 8 mM in extensor digitorumlongus (EDL; ~23°C). Ca²⁺release subsequently adapted or inactivated; this was surmounted byhigher doses. Motion also adapted but was not surmounted. Prolonged exposure to caffeine evidently suppressed myofilament interaction inboth types of fiber. In EDL fibers, 16 mM caffeine moderately increasedlocal Ca²⁺ transients. In soleusfibers, 16 mM caffeine greatly increased Ca²⁺ release and producedpropagated waves of Ca²⁺(~1.5-2.5 µm/s). Ca²⁺waves in slow-twitch fibers reflect the caffeine-sensitive mechanism ofCa²⁺-inducedCa²⁺ release. Fast-twitch fiberspossibly lack this mechanism, which could account for their lowersensitivity to caffeine.